Bifunctional Molecules for Lysosomal Targeting and Related Compositions and Methods

ABSTRACT

Provided are bifunctional molecules that include a first moiety that specifically binds a cell surface molecule or extracellular molecule, and a second moiety that specifically binds a lysosomal targeting molecule. The bifunctional molecules find use, e.g., for targeted degradation of cell surface and extracellular molecules (e.g., proteins) via the endosomal/lysosomal pathway. Also provided are compositions and kits that include the bifunctional molecules, as well as methods of using the bifunctional molecules. Methods of making bifunctional molecules are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/782,193, filed Dec. 19, 2018, and U.S. ProvisionalPatent Application No. 62/932,347, filed Nov. 7, 2019, whichapplications are incorporated herein by reference in their entireties.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under contractsCA227942, GM059907, and GM123636 awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

SUMMARY

Provided are bifunctional molecules that include a first moiety thatspecifically binds a cell surface molecule or extracellular molecule,and a second moiety that specifically binds a lysosomal targetingmolecule. The bifunctional molecules find use, e.g., for targeteddegradation of cell surface and extracellular molecules (e.g., proteins)via the endosomal/lysosomal pathway. Also provided are compositions andkits that include the bifunctional molecules, as well as methods ofusing the bifunctional molecules. Methods of making bifunctionalmolecules are also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 A schematic illustration of a bifunctional molecule and usethereof according to one embodiment of the present disclosure.

FIG. 2 A scheme for synthesizing mannose-6-phosphate N-carboxyanhydrideaccording to one embodiment. This route allows access tomannose-6-phosphate glycans linked to, e.g., serine residues for use asmonomers in N-carboxyanhydride polymerization.

FIG. 3 A scheme for synthesizing mannose-6-phosphonateN-carboxyanhydride according to one embodiment. This route allows accessto mannose-6-phosphonate glycans linked to, e.g., serine residues foruse as monomers in N-carboxyanhydride polymerization. The phosphonategroup is a hydrolytically stable variant of a phosphate group which haspreviously demonstrated better serum stability compared tomannose-6-phosphate glycans.

FIG. 4 A scheme for synthesizing mannose-6-carboxylateN-carboxyanhydride according to one embodiment. This route allows accessto mannose-6-carboxylate glycans linked to, e.g., serine residues foruse as monomers in N-carboxyanhydride polymerization. The carboxylategroup is a hydrolytically stable variant of a phosphate group which haspreviously demonstrated better serum stability compared tomannose-6-phosphate glycans. Mannose-6-carboxylate glycans havepreviously demonstrated a relative binding affinity of 0.3 for thecation independent M6PR (CIM6PR), compared to mannose-6-phosphateglycans. The ability to chemically tune the receptor-ligand interactionallows for greater control in biological applications to diminishoff-target binding events.

FIG. 5 A scheme for synthesizing mannose-6-acrylate N-carboxyanhydrideaccording to one embodiment. This route allows access tomannose-6-acrylate glycans linked to, e.g., serine residues for use asmonomers in N-carboxyanhydride polymerization. The acrylate group is ahydrolytically stable variant of a phosphate group which has previouslydemonstrated better serum stability compared to mannose-6-phosphateglycans. Mannose-6-acrylate glycans have previously demonstrated arelative binding affinity of 0.7 for the CIM6PR, compared tomannose-6-phosphate glycans. The ability to chemically tune thereceptor-ligand interaction allows for greater control in biologicalapplications to diminish off-target binding events.

FIG. 6 A scheme for synthesizing glucose-6-phosphonateN-carboxyanhydride according to one embodiment. This route allows accessto glucose-6-phosphonate glycans linked to, e.g., serine residues foruse as monomers in N-carboxyanhydride polymerization. Theglucose-6-phosphonate residue has a significantly weaker bindingaffinity for the CIM6PR compared to mannose-containing glycans.

FIG. 7 A scheme for synthesizing mannose-6-phosphonate isothiocyanateaccording to one embodiment. This route allows access tomannose-6-phosphonate isothiocyanate (M6Pn-ITC), which can be directlyconjugated to, e.g., lysine residues in proteins. Conjugation ofmultiple M6Pn-ITC to multiple amino acids (e.g., lysines) within a givenprotein allows for multivalent presentation of M6Pn glycans.

FIG. 8 An illustration of a general NCA polymerization scheme forsynthesizing a scaffold for displaying M6P ligands according to oneembodiment. Copolymers with other amino-acid derived NCAs are readilysynthesized similarly, and provide access to numerous polymers withvaried structures and compositions bearing multiple M6P ligand residues.These materials are subsequently deprotected to provide the fullpolypeptide/glycan structure.

FIG. 9 A scheme for solid-phase peptide synthesis ofmannose-6-phosphonate peptide oligomer according to one embodiment. Asshown, a scaffold for displaying M6P ligands can also be synthesizedusing solid-phase peptide synthesis, starting from M6P, M6Pn, etc. aminoacids. This synthetic route allows for greater control over polypeptidelength and composition compared to the NCA polymerization route, anddoes not require special synthesis conditions compared to the NCApolymerization-derived materials.

FIG. 10 A schematic illustration (top) and fluorescence imaging results(bottom) of an experiment demonstrating that M6Pn polymers which havebeen functionalized with a biotin cap can mediate transfer ofextracellular NeutrAvidin-647 (NA647—a protein to which biotin stronglybinds) to lysosomes from the extracellular space for degradation.Colocalization of both protein and lysosome staining dye are observed.

FIG. 11 Data demonstrating that several cell lines exhibit uptake ofNA647 in a M6Pn polymer dependent manner. In view of these results, itis expected that any cell line bearing M6PRs (e.g., CIM6PRs) will allowfor shuttling of proteins to lysosome by this method, and is not limitedto the cell lines tested in the present study.

FIG. 12 Data demonstrating that native gel polyacrylamide gelelectrophoresis (PAGE) can be used to monitor functionalization ofproteins (including antibodies) by M6P polymers. In this example,functionalizations were carried out by first labeling the protein ofinterested with a reactive alkyne (e.g., bicyclo[6.1.0]nonyne, BCN),followed by incubation with an azide-containing polymer (abio-orthogonal copper-free strain-promoted click reaction).

FIG. 13 Schematic illustration (top) and fluorescence imaging data(bottom) demonstrating that poly(M6Pn) labeled antibodies can shuttletheir binding partners to lysosomes. In this example, a mouse IgG-488was incubated with an anti-mouse IgG antibody bearing the poly(M6Pn)tag, with colocalization of both protein and lysosome staining dye(merge).

FIG. 14 Schematic illustration (top) and data (bottom) demonstratingthat poly(M6Pn) labeled antibodies can shuttle their binding partners tointracellular compartments. In this example, recombinant human apoE4 wasincubated with a mouse-derived anti-human apoE4 antibody, an anti-mouseIgG antibody, or an anti-mouse antibody bearing the poly(M6Pn) tag.Significantly more uptake is observed with the M6Pn-containing secondaryantibody.

FIG. 15 Data demonstrating that poly(M6Pn) labeled antibodies canshuttle their binding partners for degradation. In this example, EGFRdegradation was assessed by incubating cells with cetuximab bearing anM6Pn tag. Loss of total EGFR is observed for all cell lines tested,compared to cetuximab or cetuximab bearing a mock polymer (GalNAc). EGFis a positive control for EGFR degradation. Lane 1: control. Lane 2: EGF(100 ng/mL, 1 h, +control). Lane 3: cetuximab. Lane 4: cetuximab-GalNAcconjugate. Lane 5: cetuximab-M6P conjugate (long). Lane 6: cetuximab-M6Pconjugate (short). Percent of control was calculated by densitometry.

FIG. 16 Data demonstrating that poly(M6Pn) labeled antibody fragmentscan shuttle their binding partners to lysosomes. In this example, EGFRdegradation was assessed by incubating cells with cetuximab-derived Fabportions bearing an M6Pn tag. Loss of total EGFR is observed compared tocetuximab Fab alone or cetuximab Fab bearing a mock polymer (GalNAc).

FIG. 17 Data demonstrating that poly(M6Pn) labeled antibodies canshuttle their binding partners for degradation. In this example,degradation of CD71 (transferrin receptor) was assessed by incubatingcells with a primary mouse-derived antibody against CD71, an anti-mouseIgG antibody, or an anti-mouse antibody bearing the poly(M6Pn) tag. Thesystem containing the M6P tag leads to significantly more degradation.

FIG. 18 Data demonstrating that poly(M6Pn) labeled antibodies fragmentscan shuttle their binding partners for degradation. In this example,PDL1 degradation was assessed by incubating cells with anti-PDL1antibody or anti-PDL1 antibody bearing an M6P tag. Degradation is onlyobserved with M6P-labeled anti-PDL1 antibody.

FIG. 19 Schematic illustration of targeted extracellular proteindegradation using a bifunctional molecule which is a bispecificantibody. In this example, a bispecific antibody against the CIM6PR anda given target, which disengages from the target at the lowered pH ofthe endosomes. This strategy allows for a given bispecific antibody tocycle with the receptor and continuously delivery cargo and targets tothe lysosome, without degradation of the antibody.

FIG. 20 Schematic illustration of a lysosomal targeting molecule towhich the second moiety of a bifunctional molecule may bind according toembodiments of the present disclosure. In this example, the lysosomaltargeting molecule is asialoglycoprotein receptor (ASGPR) which isexpressed substantially exclusively on liver cells, e.g., hepatocytes.As shown on the right, ASGPR constitutively recycles between the plasmamembrane and the endosome, thereby bringing extracellular glycoproteinsinside the cell for degradation in the lysosome. Shown on the lower leftis an example bifunctional molecule comprising a first moiety that is anantibody (e.g., an antibody that binds a molecule expressed on thesurface of hepatocytes or a molecule present in the extracellular spaceof hepatocytes) and a second moiety comprising multivalent ASGPR ligandsfor binding to ASGPR. In this example, the second moiety comprises apolymer of N-acetylgalactosamines (GalNAc), in particular apoly(GalNAc-co-Ala) polymer as shown.

FIG. 21 Schematic illustration and data demonstrating import of targetmolecules into HEPG2 (hepatocellular carcinoma) cells using abifunctional molecule comprising a first moiety (in this example, anantibody) that binds to the target molecule and a second moietycomprising the GalNAc-containing polymer shown in FIG. 20.

FIG. 22 Schematic illustration and data demonstrating efficient cellularuptake via ASGPR in HUH7 (hepatocellular carcinoma) cells.

FIG. 23 Schematic illustration and data demonstrating efficientdegradation of EGFR in HEP3B (hepatocellular carcinoma) cells using abifunctional molecule comprising Cetuximab (first moiety) conjugated toa second moiety comprising the GalNAc-containing polymer shown in FIG.20. Also shown is EGFR degradation data for a conjugate comprisingCetuximab (first moiety) conjugated to a second moiety comprising M6PRligands.

FIG. 24 Data demonstrating efficient degradation of EGFR in HEPG2 cellsusing a bifunctional molecule comprising Cetuximab (first moiety)conjugated to a second moiety comprising the GalNAc-containing polymershown in FIG. 20. Also shown is EGFR degradation data for a conjugatecomprising Cetuximab (first moiety) conjugated to a second moietycomprising M6PR ligands.

FIG. 25 Data from a time-course study assessing EGFR degradation overtime in HEP3B cells.

FIG. 26 Immunofluorescence data demonstrating that treatment of HEP3Bcells using the Cetuximab conjugates described above degrades themajority of membrane EGFR, whereas residual EGFR is internal to thecells.

FIG. 27 Western blot data showing the extent of degradation of HER2 inHUH7 and HEPG2 cells in the presence of Trastuzumab alone or Trastuzumabconjugated to a GalNAc-containing polymer.

DETAILED DESCRIPTION

Provided are bifunctional molecules that include a first moiety thatspecifically binds a cell surface molecule or extracellular molecule,and a second moiety that specifically binds a lysosomal targetingmolecule. The bifunctional molecules find use, e.g., for targeteddegradation of cell surface and extracellular molecules (e.g., proteins)via the endosomal/lysosomal pathway. Also provided are compositions andkits that include the bifunctional molecules, as well as methods ofusing the bifunctional molecules. Methods of making bifunctionalmolecules are also provided.

Before the bifunctional molecules, compositions, kits and methods of thepresent disclosure are described in greater detail, it is to beunderstood that the bifunctional molecules, compositions, kits andmethods are not limited to particular embodiments described, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting, since the scope of thebifunctional molecules, compositions, kits and methods will be limitedonly by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the bifunctional molecules,compositions, kits and methods. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges andare also encompassed within the bifunctional molecules, compositions,kits and methods, subject to any specifically excluded limit in thestated range. Where the stated range includes one or both of the limits,ranges excluding either or both of those included limits are alsoincluded in the bifunctional molecules, compositions, kits and methods.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the bifunctional molecules, compositions, kits andmethods belong. Although any bifunctional molecules, compositions, kitsand methods similar or equivalent to those described herein can also beused in the practice or testing of the bifunctional molecules,compositions, kits and methods, representative illustrative bifunctionalmolecules, compositions, kits and methods are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the materials and/or methods in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present bifunctional molecules, compositions, kitsand methods are not entitled to antedate such publication, as the dateof publication provided may be different from the actual publicationdate which may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the bifunctional molecules,compositions, kits and methods, which are, for clarity, described in thecontext of separate embodiments, may also be provided in combination ina single embodiment. Conversely, various features of the bifunctionalmolecules, compositions, kits and methods, which are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any suitable sub-combination. All combinations of theembodiments are specifically embraced by the present disclosure and aredisclosed herein just as if each and every combination was individuallyand explicitly disclosed, to the extent that such combinations embraceoperable processes and/or compositions. In addition, allsub-combinations listed in the embodiments describing such variables arealso specifically embraced by the present bifunctional molecules,compositions, kits and methods and are disclosed herein just as if eachand every such sub-combination was individually and explicitly disclosedherein.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentmethods. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Bifunctional Molecules

The present disclosure provides bifunctional molecules that include afirst moiety that specifically binds a cell surface molecule orextracellular molecule, and a second moiety that specifically binds alysosomal targeting molecule. Certain non-limiting embodiments of thebifunctional molecules will now be described.

As summarized above, the bifunctional molecules of the presentdisclosure include a first moiety that specifically binds a cell surfacemolecule or extracellular molecule. In some embodiments, the firstmoiety specifically binds a cell surface molecule. By “cell surfacemolecule” is meant a molecule associated with a cell membrane, e.g.,because the molecule has a domain that inserts into or spans a cellmembrane, e.g., a cell membrane-tethering domain or a transmembranedomain. The cell surface molecule may be any cell surface molecule whichis desired for targeted degradation via the endosomal/lysosomal pathway.In some embodiments, the cell surface molecule is a cell surfacereceptor. Cell surface receptors of interest include, but are notlimited to, stem cell receptors, immune cell receptors, growth factorreceptors, cytokine receptors, hormone receptors, receptor tyrosinekinases, a receptor in the epidermal growth factor receptor (EGFR)family (e.g., HER2 (human epidermal growth factor receptor 2), etc.), areceptor in the fibroblast growth factor receptor (FGFR) family, areceptor in the vascular endothelial growth factor receptor (VEGFR)family, a receptor in the platelet derived growth factor receptor(PDGFR) family, a receptor in the rearranged during transfection (RET)receptor family, a receptor in the Eph receptor family, a receptor inthe discoidin domain receptor (DDR) family, and a mucin protein (e.g.,MUC1). In some embodiments, the cell surface molecule is CD71(transferrin receptor). In certain aspects, the cell surface receptor isan immune cell receptor selected from a T cell receptor, a B cellreceptor, a natural killer (NK) cell receptor, a macrophage receptor, amonocyte receptor, a neutrophil receptor, a dendritic cell receptor, amast cell receptor, a basophil receptor, and an eosinophil receptor.

In some embodiments, the first moiety specifically binds a cell surfacemolecule which mediates its effect not through a specific molecularinteraction (and therefore is not susceptible to blocking), but ratherthrough bulk biophysical or aggregate effects. A non-limiting example ofsuch a cell surface molecule is a mucin. Examples of mucins include, butare not limited to, MUC1, MUC16, MUC2, MUC5AC, MUC4, CD43, CD45, GPIb,and the like.

In some embodiments, when the first moiety specifically binds a cellsurface molecule, the cell surface molecule is present on a cancer cell.By “cancer cell” is meant a cell exhibiting a neoplastic cellularphenotype, which may be characterized by one or more of, for example,abnormal cell growth, abnormal cellular proliferation, loss of densitydependent growth inhibition, anchorage-independent growth potential,ability to promote tumor growth and/or development in animmunocompromised non-human animal model, and/or any appropriateindicator of cellular transformation. “Cancer cell” may be usedinterchangeably herein with “tumor cell”, “malignant cell” or “cancerouscell”, and encompasses cancer cells of a solid tumor, a semi-solidtumor, a hematological malignancy (e.g., a leukemia cell, a lymphomacell, a myeloma cell, etc.), a primary tumor, a metastatic tumor, andthe like. In some embodiments, the cell surface molecule present on thecancer cell is a tumor-associated antigen or a tumor-specific antigen.

In certain aspects, when the first moiety specifically binds a cellsurface molecule, the cell surface molecule is present on an immunecell. In some embodiments, the cell surface molecule is present on animmune cell selected from a T cell, a B cell, a natural killer (NK)cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a mastcell, a basophil, and an eosinophil. In certain aspects, the cellsurface molecule present on the immune cell is an inhibitory immunereceptor. As used herein, an “inhibitory immune receptor” is a receptorpresent on an immune cell that negatively regulates an immune response.Examples of inhibitory immune receptors which may be inhibited accordingto the methods of the present disclosure include inhibitory immunereceptors of the Ig superfamily, including but not limited to: CD200R,CD300a (IRp60; mouse MAIR-1), CD300f (IREM-1), CEACAM1 (CD66a), FcγRIIb,ILT-2 (LIR-1; LILRB1; CD85j), ILT-3 (LIR-5; CD85k; LILRB4), ILT-4(LIR-2; LILRB2), ILT-5 (LIR-3; LILRB3; mouse PIR-B); LAIR-1, PECAM-1(CD31), PILR-α (FDF03), SIRL-1, and SIRP-α. Further examples ofinhibitory immune receptors which may be inhibited according to themethods of the present disclosure include sialic acid-binding Ig-likelectin (Siglec) receptors, e.g., Siglec 7, Siglec 9, and/or the like.Additional examples of inhibitory immune receptors which may beinhibited according to the methods of the present disclosure includeC-type lectins, including but not limited to: CLEC4A (DCIR), Ly49Q andMICL. Details regarding inhibitory immune receptors may be found, e.g.,in Steevels et al. (2011) Eur. J. Immunol. 41(3):575-587. In someembodiments, the cell surface molecule present on the immune cell is aligand of an inhibitory immune receptor. In certain aspects, the cellsurface molecule present on the immune cell is an immune checkpointmolecule. Non-limiting examples of immune checkpoint molecules to whichthe first moiety may specifically bind include PD-1, PD-L1, CTLA4, TIM3,LAG3, TIGIT, and a member of the B7 family.

As summarized above, the bifunctional molecules of the presentdisclosure include a first moiety that specifically binds a cell surfacemolecule or extracellular molecule. In some embodiments, the firstmoiety specifically binds an extracellular molecule. By “extracellularmolecule” is meant a soluble molecule external to the cell membranes ofany cells in the vicinity of the soluble molecule. The extracellularmolecule may be any extracellular molecule which is desired for targeteddegradation via the endosomal/lysosomal pathway. In some embodiments,the extracellular molecule is a ligand for a cell surface receptor. Cellsurface receptor ligands of interest include, but are not limited to,growth factors (e.g., epidermal growth factor (EGF), vascularendothelial growth factor (VEGF), and the like), cytokines (e.g., aninterleukin, an interferon, a tumor necrosis factor (TNF), atransforming growth factor β (TGF-β), including any particular subtypesof such cytokines), hormones, and the like. In certain aspects, thefirst moiety specifically binds apolipoprotein E4 (ApoE4).

In some embodiments, the first moiety specifically binds anextracellular molecule, where the extracellular molecule is an antibody,e.g., an antibody that specifically binds a cell surface molecule ordifferent extracellular molecule. In some embodiments, the antibody isan autoantibody. Non-limiting examples of autoantibodies includerheumatoid factor (RF), antinuclear antibody (ANA), AntineutrophilCytoplasmic Antibodies (ANCA), Anti-Double Stranded DNA (anti-dsDNA),Anticentromere Antibodies (ACA), Antihistone Antibodies, CyclicCitrullinated Peptide Antibodies (CCP), Extractable Nuclear AntigenAntibodies (e.g., anti-SS-A (Ro) and anti-SS-B (La), anti-RNP,anti-Jo-1, anti-Sm, Scl-70), Cardiolipin Antibodies, Beta-2 Glycoprotein1 Antibodies, Antiphospholipid Antibodies (APA), Lupus anticoagulants(LA), Diabetes-related Autoantibodies, Anti-Tissue Transglutaminase(anti-tTG), Anti-Gliadin Antibodies (AGA), Intrinsic Factor Antibodies,Parietal Cell Antibodies, Thyroid Autoantibodies (e.g., anti-TPO, TSHreceptor antibodies), Smooth Muscle Antibodies (SMA), AntimitochondrialAntibodies (AMA), Liver Kidney Microsome Type 1 Antibodies (anti-LKM-1),Anti-Glomerular Basement Membrane (GBM), Acetylcholine Receptor (AChR)Antibodies, etc.

In some embodiments, the first moiety specifically binds anextracellular molecule, where the extracellular molecule is a secretedprotein that accumulates in disease (e.g., alpha-synuclein), acholesterol carrier (e.g., ApoB), an infectious disease toxin (e.g., ABtoxins, ESAT-6), an infectious particle (e.g., a whole virus, a wholebacterium, etc.), a clotting factor (e.g., Factor IX), the target of anyFDA approved antibody that binds to an extracellular molecule (e.g.,TNFalpha), any chemokine or cytokine (e.g., mediators of sepsis orchronic inflammation such at IL-1), a proteinaceous hormone (e.g.,insulin, ACTH, etc.), a proteinaceous mediator of a mood disorder, aproteinaceous mediator of energy homeostasis (e.g., leptin, ghrelin,etc.), a proteinaceous allergen present in the bloodstream or anantibody against such an allergen (e.g., for peanut allergies), aproteinaceous toxin (e.g., snake venom hyaluronidase, etc.), etc.

In certain embodiments, the first moiety specifically binds a cellsurface or extracellular molecule, where the cell surface orextracellular molecule is a mutated protein. In some embodiments, thebifunctional molecule causes shuttling of the mutated protein into thelysosome, promoting its loading onto a major histocompatibility complex(MHC) (e.g., MHC I or MHC II), and thereby promoting recognition of themutated protein by the immune system. In this context, the bifunctionalmolecule finds use in generating antibodies specific to a mutated andunwanted protein (e.g., KIT).

By “specifically binds” is meant the first moiety and the second moietybind to their respective targets with an affinity or K_(a) (that is, anequilibrium association constant of a particular binding interactionwith units of 1/M) of, for example, greater than or equal to about 105M⁻¹. In certain embodiments, the first moiety and the second moiety bindto their respective targets with a K_(a) greater than or equal to about10⁶ M⁻¹, 107 M⁻¹, 10⁸ M⁻¹, 109 M⁻¹, 10¹⁰ M⁻¹, 10¹¹ M⁻¹, 10¹² M⁻¹, or10¹³ M⁻¹. “High affinity” binding refers to binding with a K_(a) of atleast 107 M⁻¹, at least 10⁸ M⁻¹, at least 109 M⁻¹, at least 10¹⁰ M⁻¹, atleast 10¹¹ M⁻¹, at least 10¹² M⁻¹, at least 10¹³ M⁻¹, or greater.Alternatively, affinity may be defined as an equilibrium dissociationconstant (K_(D)) of a particular binding interaction with units of M(e.g., 10⁻⁵ M to 10⁻¹³ M, or less). In certain aspects, specific bindingmeans the first moiety and the second moiety bind to their respectivetargets with a K_(D) of less than or equal to about 10⁻⁵ M, less than orequal to about 10⁻⁶ M, less than or equal to about 10⁻⁷ M, less than orequal to about 10⁻⁸ M, or less than or equal to about 10⁻⁹ M, 10⁻¹⁰ M,10⁻¹¹ M, or 10⁻¹² M or less. The binding affinity of the first moietyand the second moiety to their respective targets can be readilydetermined using conventional techniques, e.g., by competitive ELISA(enzyme-linked immunosorbent assay), equilibrium dialysis, by usingsurface plasmon resonance (SPR) technology (e.g., the BIAcore 2000instrument, using general procedures outlined by the manufacturer); byradioimmunoassay; or the like.

The first moiety may be any type moiety capable of binding to the cellsurface molecule or extracellular molecule to be targeted fordegradation via the endosomal/lysosomal pathway. In certain aspects, thefirst moiety is selected from a polypeptide, a ligand (e.g., a ligandfor a cell surface receptor, where the cell surface receptor is targetedfor degradation), an aptamer, a nanoparticle, and a small molecule. Thesecond moiety may be any type moiety capable of binding to the lysosomaltargeting molecule. In certain aspects, the second moiety is selectedfrom a polypeptide, a ligand (e.g., a ligand for the lysosomal targetingmolecule), an aptamer, a nanoparticle, and a small molecule.

In some embodiments, when a moiety of the bifunctional molecule is apolypeptide, the moiety is an antibody. The terms “antibody” and“immunoglobulin” include antibodies or immunoglobulins of any isotype(e.g., IgG (e.g., IgG1, IgG2, IgG3 or IgG4), IgE, IgD, IgA, IgM, etc.),whole antibodies (e.g., antibodies composed of a tetramer which in turnis composed of two dimers of a heavy and light chain polypeptide);single chain antibodies; fragments of antibodies (e.g., fragments ofwhole or single chain antibodies) which retain specific binding to thecell surface molecule or extracellular molecule (in the case of thefirst moiety) or lysosomal targeting molecule (in the case of the secondmoiety), including, but not limited to, Fv, single chain Fv (scFv), Fab,F(ab′)₂, Fab′, (scFv′)₂, diabodies, and nanobodies; chimeric antibodies;monoclonal antibodies; fully human antibodies; humanized antibodies(e.g., humanized whole antibodies, humanized antibody fragments, etc.);and fusion proteins including an antigen-binding portion of an antibodyand a non-antibody protein or fragment thereof. The antibodies may bedetectably labeled, e.g., with an in vivo imaging agent, or the like.The antibodies may be further conjugated to other moieties, such as,e.g., polyethylene glycol (PEG), etc. Fusion to an antibody Fc region(or a fragment thereof), conjugation to PEG, etc. may find use, e.g.,for increasing serum half-life of the antibody upon administration tothe subject.

By “small molecule” is meant a compound having a molecular weight of1000 atomic mass units (amu) or less. In some embodiments, the smallmolecule is 750 amu or less, 500 amu or less, 400 amu or less, 300 amuor less, or 200 amu or less. In certain aspects, the small molecule isnot made of repeating molecular units such as are present in a polymer.

As summarized above, the second moiety specifically binds a lysosomaltargeting molecule. As used herein, a “lysosomal targeting molecule” isa cell surface molecule which, upon being bound by the second moiety ofthe bifunctional molecule, shuttles the bifunctional molecule and cellsurface molecule or extracellular molecule bound by the first moiety tothe lysosome within the cell. Upon delivery and internalization into thelysosome, the bifunctional molecule and cell surface molecule orextracellular molecule are degraded by lysosomal enzymes, e.g., acidhydrolases. In this way, the bifunctional molecule targets the cellsurface molecule or extracellular molecule bound by the first moiety fordegradation, which targeting finds use in a variety of in vitro and invivo applications, including research and clinical applications.

The second moiety may bind to any suitable lysosomal targeting molecule.Non-limiting examples of lysosomal targeting molecules include amannose-6-phosphate receptor (M6PR), sortilin, folate receptor, ASPGR,IFITM3, molecules in the endosome/lysosome pathway (e.g., LIMP-1,LIMP-2), etc.

In some embodiments, the lysosomal targeting molecule to which thesecond moiety binds is a mannose-6-phosphate receptor (M6PR). M6PRs arepresent throughout the tissues of the body and are responsible fortrafficking mannose-6-phosphate (M6P)-tagged cargo, such as acidhydrolases, from the Golgi compartment and extracellular space to thelysosome. Details regarding M6PRs may be found, e.g., in Gary-Bobo etal. (2007) Curr. Med. Chem. 14:2945-2953; Das et al. (2016) ACS MacroLett. 5:809-813; and elsewhere. Examples of M6PRs to which the secondmoiety may bind include the cation-dependent human M6PR provided asUniProtKB-P20645 and the cation-independent M6PR provided asUniProtKB-P11717 (also referred to as insulin-like growth factor 2receptor (IGF2R)). The cation-independent mannose 6-phosphate receptoris a multifunctional protein which binds at the cell surface to ligandssuch as mannose 6-phosphate (M6P) bearing proteins, IGF-II, retinoicacid, plasminogen, etc. Its major function is to bind and transportM6P-enzymes to lysosomes, but it can also modulate the activity of avariety of extracellular M6P-glycoproteins, e.g., latent TGFβ precursor,urokinase-type plasminogen activator receptor, granzyme B, growthfactors, herpes virus, etc.

In certain aspects, when the lysosomal targeting molecule is an M6PR,the second moiety is an antibody that specifically binds the M6PR.Anti-M6PR antibodies are available and include the MOB-1772z recombinantanti-human M6PR antibody (Creative Biolabs), the EPR6599, 2G11, MEM-238,EPR6599, and EPR7691 anti-M6PR antibodies (Abcam), and the like.

In some embodiments, when the lysosomal targeting molecule is an M6PR,the second moiety includes one or more M6PR ligands. In certain aspects,the one or more M6PR ligands include one or more mannose-6-phosphates(M6Ps), where M6P has the following structure:

Alternatively, or additionally, the one or more M6PR ligands include oneor more M6P analogs. By “M6P analog” is meant a molecule that is not M6Pbut binds to an M6P recognition site of M6PR. Several M6P analogs withphosphonate, carboxylate, sulfate, sulfonate or malonate groups displaya higher affinity and a stronger stability in human serum than M6Pitself. Some structural features have been shown to be important for thebinding of M6P to M6PR. For example, the hydroxyl group at the2-position of the pyranose ring is axial, strong binding to M6PR isobserved. The distance between the negative charge and the pyranose ringalso plays a role in M6P recognition by M6PR. It has been shown thatsuitable analogs: should generally be isosteric to M6P to efficientlybind M6PR; a single negative charge is sufficient to allow binding toM6PR while the phosphorus atom is not necessary to ensure recognition;and the presence of two negative charges (as in the malonate andphosphonate isosteric analogs of M6P) is beneficial for binding to M6PR.In some embodiments, when the one or more M6PR ligands include one ormore M6PR analogs, the one or more M6PR analogs include one or morephosphonate M6P analogs (M6Pn), malonate M6P analogs, carboxylate M6Panalogs, sulfonate M6P analogs, acrylate M6P analogs, and/or the like.In some embodiments, the one or more M6PR analogs include one or morephosphonate M6P analogs (M6Pn) having the structure (where M⁺ is anycountercation or hydrogen atom):

In some embodiments, the one or more M6PR analogs include one or morecarboxylate M6P analogs having one of the following structures (where M⁺is any countercation or hydrogen atom):

In some embodiments, the one or more M6PR analogs include one or moremalonate M6P analogs having the following structure (where M⁺ is anycountercation or hydrogen atom):

Details regarding M6P and M6P analog recognition by M6PR, as well as M6Panalogs that may be employed in the bifunctional molecules of thepresent disclosure may be found, e.g., in Gary-Bobo et al. (2007) Curr.Med. Chem. 14:2945-2953; and Jeanjean et al. (2008) Bioorg Med ChemLett. 18(23): 6240-3, the disclosures of which are incorporated hereinby reference in their entireties for all purposes.

In certain aspects, when the second moiety includes one or more M6PRligands, the second moiety includes from 1 to 1000 M6PR ligands, such asfrom 1 to 750, 1 to 500, 1 to 250, 1 to 100, 1 to 75, 1 to 50, 1 to 40,1 to 30, 1 to 20, 1 to 10 (e.g., 1 to 6), or 1 to 5 M6PR ligands. Insome embodiments, when the second moiety includes one or more M6PRligands, the second moiety includes from 10 to 50, 15 to 45, 20 to 40,or 25 to 35 M6PR ligands. In certain aspects, when the second moietyincludes one or more M6PR ligands, the second moiety includes 5 or more,10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 75 or more,100 or more, 250 or more, 500 or more, 750 or more, or 1000 or more M6PRligands.

In some embodiments, when the second moiety includes one or more M6PRligands, the second moiety includes a polymer scaffold that displays(e.g., is functionalized with) the one or more M6PR ligands. One exampleof such a bifunctional molecule and use thereof is schematicallyillustrated in FIG. 1. In this example, the bifunctional moleculeincludes an antibody as the first moiety. The antibody may bind either atarget extracellular molecule (as shown on the left) or a target cellsurface molecule (as shown on the right). The antibody is conjugated toa polymer scaffold that displays M6P ligands (each of which isdesignated “6P” in FIG. 1). Upon binding of a displayed M6P ligand by acell surface M6PR, the M6PR shuttles the bifunctional molecule (andbound target molecule) to the lysosome for degradation.

When the second moiety includes a polymer scaffold that displays the oneor more M6PR ligands, the polymer scaffold may be a glycopolymerincluding the one or more M6PR ligands. By way of example, theglycopolymer may be a glycoprotein including one or more amino acids(e.g., natural and/or non-natural amino acids) functionalized with theone or more M6PR ligands. When the glycopolymer is a glycoprotein, theglycoprotein may be a N-carboxyanhydride (NCA)-derived glycoprotein. Thering-opening polymerization (ROP) of NCA monomers is a well-studiedroute to synthetic polypeptides and polypeptide hybrids that possess abroad range of useful physical properties. In some embodiments, thepolymerization is metal-catalyzed. Suitable approaches for large-scalesynthesis of α-amino acid-N-carboxyanhydrides are described, e.g., inSemple et al. (2016) Synthetic Communications 47(1):53-61.

An example approach for synthesizing mannose-6-phosphateN-carboxyanhydride is schematically illustrated in FIG. 2. An exampleapproach for synthesizing mannose-6-phosphonate N-carboxyanhydride isschematically illustrated in FIG. 3. An example approach forsynthesizing mannose-6-carboxylate N-carboxyanhydride is schematicallyillustrated in FIG. 4. An example approach for synthesizingmannose-6-acrylate N-carboxyanhydride is schematically illustrated inFIG. 5. An example approach for synthesizing glucose-6-phosphonateN-carboxyanhydride is schematically illustrated in FIG. 6. An exampleapproach for synthesizing mannose-6-phosphonate isothiocyanate isschematically illustrated in FIG. 7.

In certain embodiments, a bifunctional molecule of the presentdisclosure comprises a second moiety that specifically binds a lysosomaltargeting molecule expressed on the surface of liver cells, e.g.,hepatocytes (including hepatocellular carcinoma (HCC) cells). Anon-limiting example of a lysosomal targeting molecule expressed on thesurface of liver cells to which the second moiety may bind isasialoglycoprotein receptor (ASGPR)—a hepatic receptor that mediatesremoval of glycoconjugates from blood. The receptor comprises twoproteins, asialoglycoprotein receptor 1 and 2 (ASGR1(UniProtKB-P07306—human) and ASGR2 (UniProtKB-P07307—human)), encoded bythe genes ASGR1 and ASGR2. ASGPR binds asialoglycoproteins, which areglycoproteins from which a sialic acid has been removed to exposegalactose and galactosamine residues. The receptors, which are locatedon liver cells, remove the target glycoproteins from circulation. ASGPRis highly expressed on the surface of hepatocytes, several humancarcinoma cell lines, and liver cancers.

When the lysosomal targeting molecule is ASGPR, suitable second moietiesinclude but are not limited to anti-ASGPR antibodies, ASGPR ligands, andthe like. According to some embodiments, such a second moiety comprisesone or more ASGPR ligands. Suitable ASGPR ligands include, but are notlimited to, one or more N-acetylgalactosamines (GalNAc), one or moregalactoses, one or more glucoses, and any combination thereof. Incertain embodiments, such a second moiety comprises from 1 to 1000 ASGPRligands, such as from 1 to 750, 1 to 500, 1 to 250, 1 to 100, 1 to 75, 1to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10 (e.g., 1 to 6), or 1 to 5ASGPR ligands. In some embodiments, when the second moiety includes oneor more ASGPR ligands, the second moiety includes from 10 to 50, 15 to45, 20 to 40, or 25 to 35 ASGPR ligands. In certain embodiments, whenthe second moiety includes one or more ASGPR ligands, the second moietyincludes 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 ormore, 50 or more, 75 or more, 100 or more, 250 or more, 500 or more, 750or more, or 1000 or more ASGPR ligands.

According to some embodiments, when the lysosomal targeting molecule isASGPR and the second moiety comprises one or more ASGPR ligands, thesecond moiety comprises a scaffold comprising the one or more ASGPRligands. In one non-limiting example, the second moiety comprises apolymer comprising GalNAc. In certain embodiments, such a second moietycomprises poly(GalNAc-co-Ala), the structure of which is provided belowand in FIG. 20.

In certain embodiments, when the lysosomal targeting molecule is ASGPRand the second moiety comprises one or more ASGPR ligands, the secondmoiety comprises a dendrimer scaffold comprising 1 (monovalent), 2(bivalent), 3 (trivalent) or 4 or more ASGPR ligands, e.g., ASGPRligands independently selected from GalNAc, galactose, and glucose. Forexample, according to some embodiments, the second moiety comprises amonovalent, bivalent, or trivalent GalNAc-containing dendrimer scaffold.A non-limiting example of a trivalent GalNAc-containing dendrimerscaffold that may be employed is the following (designated herein asTri-GalNAc dendrimer):

According to some embodiments, the second moiety comprises a monovalent,bivalent, or trivalent galactose-containing dendrimer scaffold. Anon-limiting example of a trivalent galactose-containing dendrimerscaffold that may be employed is the following (designated herein asTri-Gal dendrimer):

According to some embodiments, when the second moiety specifically bindsa lysosomal targeting molecule expressed on the surface of liver cells,e.g., ASGPR, the first moiety specifically binds to a cell surfacemolecule expressed on hepatocytes (including hepatocellular carcinoma(HCC) cells). Non-limiting examples of such cell surface moleculesinclude growth factor receptors. Growth factor receptors of interestinclude, but are not limited to, epidermal growth factor receptor(EGFR), C-Met, insulin like growth factor 1 receptor (IGF1R), fibroblastgrowth factor receptor 4 (FGFR4), HER2, and platelet-derived growthfactor receptor (PDGFR).

In certain embodiments, the bifunctional molecule finds use in degradinga growth factor on the surface of hepatocellular carcinoma (HCC) cells(e.g., in vivo upon administration to an individual having HCC to treatthe HCC), where the bifunctional molecule comprises a first moiety thatspecifically binds to EGFR, C-Met, IGF1R, FGFR4 or HER2, and the secondmoiety specifically binds to ASGPR (e.g., the second moiety may comprisea scaffold (e.g., a polymer scaffold) comprising ASGPR ligands, e.g.,GalNAc, galactose, and/or glucose).

According to some embodiments, the bifunctional molecule finds use indegrading a cell surface molecule (e.g., growth factor) expressed onfibrotic liver cells (e.g., in vivo upon administration to an individualhaving fibrosis of the liver), where the bifunctional molecule comprisesa first moiety that specifically binds a cell surface or extracellularprotein that promotes fibrosis (e.g., PDGFR), and the second moietyspecifically binds to ASGPR (e.g., the second moiety may comprise ascaffold (e.g., a polymer scaffold) comprising one or more ASGPRligands, e.g., GalNAc, galactose, and/or glucose).

In certain embodiments, the bifunctional molecule enhances degradationof the cell surface molecule or extracellular molecule relative todegradation of the cell surface molecule or extracellular molecule inthe presence of the first moiety alone. According to some embodiments,the bifunctional molecule enhances degradation of the cell surfacemolecule or extracellular molecule relative to degradation of the cellsurface molecule or extracellular molecule in the presence of the firstmoiety or the second moiety alone. By “enhances degradation” in thiscontext means the cell surface molecule or extracellular molecule isdegraded in the presence of the bifunctional molecule and is notdegraded in the presence of the first moiety alone, or the presence ofthe first moiety or second moiety alone, under the same conditions; orthe cell surface molecule or extracellular molecule is degraded in thepresence of the bifunctional molecule to a greater extent than the cellsurface molecule or extracellular molecule is degraded in the presenceof the first moiety alone, or the presence of the first moiety or secondmoiety alone, under the same conditions. When the cell surface moleculeor extracellular molecule is degraded in the presence of thebifunctional molecule to a greater extent than the cell surface moleculeor extracellular molecule is degraded in the presence of the firstmoiety alone, or the presence of the first moiety or second moiety aloneunder the same conditions, the degradation may be 1.2 fold or greater,1.4 fold or greater, 1.6 fold or greater, 1.8 fold or greater, 2 fold orgreater, 2.5 fold or greater, 3 fold or greater, 3.5 fold or greater, 4fold or greater, 4.5 fold or greater, 5 fold or greater, 5.5 fold orgreater, 6 fold or greater, 6.5 fold or greater, 7 fold or greater, 7.5fold or greater, 8 fold or greater, 8.5 fold or greater, 9 fold orgreater, 9.5 fold or greater, or 10 fold or greater in the presence ofthe bifunctional molecule.

A non-limiting example in which a bifunctional molecule of the presentdisclosure enhances degradation of a cell surface molecule relative todegradation of the cell surface molecule in the presence of the firstmoiety alone is provided in Example 4 of the Experimental section below.An example of a bifunctional molecule that does not enhance degradationof a cell surface molecule relative to degradation of the cell surfacemolecule in the presence of the first moiety alone is provided inExample 5.

One of ordinary skill in the art can readily determine whether abifunctional molecule of interest enhances degradation of a cell surfacemolecule or extracellular molecule relative to degradation of the cellsurface molecule or extracellular molecule in the presence of the firstmoiety alone, or in the presence of the first moiety or second moietyalone. Non-limiting examples of suitable approaches for readily makingsuch a determination are provided in the Experimental section below.

A bifunctional molecule of the present disclosure may be in any suitableformat. In some embodiments, the first moiety is a polypeptide, thesecond moiety is a polypeptide, and the bifunctional molecule is afusion protein comprising the first moiety fused to the second moiety.The first moiety may be fused directly to the second moiety. In otheraspects, the first moiety may be fused indirectly to the second moiety,e.g., where a spacer domain is disposed between the first and secondmoieties. Also provided by the present disclosure are nucleic acids thatencode the bifunctional molecule when the bifunctional molecule is afusion protein. Expression vectors that include such nucleic acids arealso provided, as are cells (e.g., host cells) that include any of thenucleic acids and/or expression vectors of the present disclosure. Alsoprovided are methods of producing such cells, the methods includingintroducing into a cell any of the nucleic acids and/or expressionvectors of the present disclosure, e.g., using a suitable celltransfection protocol and transfection reagents. Also provided by thepresent disclosure are methods of making the bifunctional molecule whenthe bifunctional molecule is a fusion protein. Such methods may includeculturing a cell of the present disclosure under conditions in which thebifunctional molecule is expressed in the cell.

Other suitable formats for the bifunctional molecules of the presentdisclosure include conjugates. Accordingly, in some embodiments, abifunctional molecule of the present disclosure includes the firstmoiety conjugated to the second moiety. In certain aspects, the firstmoiety is an antibody and the second moiety specifically binds M6PR. Byway of example, the first moiety may be an antibody and the secondmoiety may include a polymer scaffold that includes/displays one or moreM6PR ligands, e.g., one or more M6Ps and/or M6P analogs, where thepolymer scaffold is conjugated to the antibody. In some suchembodiments, the second moiety is a glycoprotein including one or moreamino acids functionalized with the one or more M6PR ligands. Methods ofmaking such conjugates are also provided, the methods includingconjugating the first moiety to the second moiety. A non-limitingexample of such a method, where the first moiety is an antibody and thesecond moiety is a glycopolymer as described herein, is schematicallyillustrated in FIG. 12. In some embodiments, the methods includesite-specifically conjugating the first moiety to the second moiety. Forexample, when the first moiety includes a polypeptide (e.g., anantibody), the conjugating may include site-specifically conjugating thesecond moiety to a pre-selected amino acid of the first moiety. Incertain aspects, the pre-selected amino acid is at the N-terminus orC-terminus of the first moiety. In other aspects, the pre-selected aminoacid is internal to the first moiety—that is, between the N-terminal andC-terminal amino acid of the first moiety. In some embodiments, thepre-selected amino acid is a non-natural amino acid. Non-limitingexamples of non-natural amino acids which may be provided to the firstand/or second moieties to facilitate conjugation include those having afunctional group selected from an azide, alkyne, alkene, amino-oxy,hydrazine, aldehyde (e.g., formylglycine, e.g., SMARTag™ technology fromCatalent Pharma Solutions), nitrone, nitrile oxide, cyclopropene,norbornene, iso-cyanide, aryl halide, and boronic acid functional group.Unnatural amino acids which may be incorporated and selected to providea functional group of interest are known and described in, e.g., Maza etal. (2015) Bioconjug. Chem. 26(9):1884-9; Patterson et al. (2014) ACSChem. Biol. 9:592-605; Adumeau et al. (2016) Mol. Imaging Biol.(2):153-65; and elsewhere.

In some embodiments, conjugating the first moiety to the second moietyincludes conjugating the second moiety to a glycan on the first moiety,or vice versa. Such a method may include modifying one or more glycanson the first moiety to provide a functional group to which the secondmoiety may be attached. In one non-limiting example, N-glycans on thefirst moiety (e.g., an antibody) may be modified via periodate oxidationto aldehyde groups, which could then be functionalized with the secondmoiety, e.g., aminooxy M6Pn.

When the bifunctional molecule is a conjugate, one or more linkers maybe employed to facilitate conjugation of the first moiety to the secondmoiety. Non-limiting examples of such linkers include ester linkers,amide linkers, maleimide or maleimide-based linkers; valine-citrullinelinkers; hydrazone linkers; N-succinimidyl-4-(2-pyridyldithio)butyrate(SPDB) linkers;Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)linkers; vinylsulfone-based linkers; linkers that include polyethyleneglycol (PEG), such as, but not limited to tetraethylene glycol; linkersthat include propanoic acid; linkers that include caproleic acid, andlinkers including any combination thereof. In certain aspects, thelinker is a chemically-labile linker, such as an acid-cleavable linkerthat is stable at neutral pH (bloodstream pH 7.3-7.5) but undergoeshydrolysis upon internalization into the mildly acidic endosomes (pH5.0-6.5) and lysosomes (pH 4.5-5.0) of a target cell (e.g., a cancercell). Chemically-labile linkers include, but are not limited to,hydrazone-based linkers, oxime-based linkers, carbonate-based linkers,ester-based linkers, etc. According to certain embodiments, the linkeris an enzyme-labile linker, such as an enzyme-labile linker that isstable in the bloodstream but undergoes enzymatic cleavage uponinternalization into a target cell, e.g., by a lysosomal protease (suchas cathepsin or plasmin) in a lysosome of the target cell (e.g., acancer cell). Enzyme-labile linkers include, but are not limited to,linkers that include peptidic bonds, e.g., dipeptide-based linkers suchas valine-citrulline linkers, such as amaleimidocaproyl-valine-citruline-p-aminobenzyl (MC-vc-PAB) linker, avalyl-alanyl-para-aminobenzyloxy (Val-Ala-PAB) linker, and the like.Chemically-labile linkers, enzyme-labile, and non-cleavable linkers areknown and described in detail, e.g., in Ducry & Stump (2010)Bioconjugate Chem. 21:5-13.

Numerous strategies are available for conjugating the first and secondmoieties through a linker. For example, the first moiety may bederivatized by covalently attaching the linker to the first moiety,where the linker has a functional group capable of reacting with a“chemical handle” on the second moiety. Also by way of example, thesecond moiety may be derivatized by covalently attaching the linker tothe second moiety, where the linker has a functional group capable ofreacting with a “chemical handle” on the first moiety. The functionalgroup on the linker may vary and may be selected based on compatibilitywith the chemical handle on the first or second moiety. According to oneembodiment, the chemical handle is provided by incorporation of anunnatural amino acid having the chemical handle into the first or secondmoiety. In some embodiments, conjugating the first and second moietiesis by alkyne-azide cycloaddition.

Other suitable formats for the bifunctional molecules of the presentdisclosure include bispecific antibodies. For example, a bifunctionalmolecule of the present disclosure may be a bispecific antibody wherethe first moiety (e.g., a first Fab arm) specifically binds a cellsurface molecule or extracellular molecule, and the second moiety (e.g.,a second Fab arm) specifically binds a lysosomal targeting molecule(e.g., M6PR). A schematic illustration of such a bispecific antibody isprovided in FIG. 19. In some embodiments, the bispecific antibodydisengages from the target at the lowered pH of the endosomes. Such astrategy allows for a given bispecific antibody to cycle with thereceptor and continuously deliver cargo and targets to the lysosome,without degradation of the antibody. Approaches for making bispecificantibodies are known. For example, when the bifunctional molecule is abispecific antibody, the bispecific antibody may be made using a“knobs-into-holes” (KIHs) approach. KIHs technology involves engineeringCH3 domains to create either a “knob” or a “hole” in each heavy chain topromote heterodimerization. KIHs design and production strategies areknown and include those described, e.g., in Xu et al. (2015) MAbs7(1):231-42; Carter et al. (2001) J. Immunol. Methods 248(1-2):7-15;Ridgway et al. (1996) Protein Eng. 9(7):617-2; and Merchant et al.(1998) Nat. Biotechnol. 16(7):677-81.

Compositions

As summarized above, the present disclosure provides compositions. Thecompositions may include any of the bifunctional molecules of thepresent disclosure, including any of the bifunctional moleculesdescribed in the Bifunctional Molecule section above, which isincorporated but not reiterated herein for purposes of brevity.

In certain aspects, the compositions include a bifunctional molecule ofthe present disclosure present in a liquid medium. The liquid medium maybe an aqueous liquid medium, such as water, a buffered solution, and thelike. One or more additives such as a salt (e.g., NaCl, MgCl₂, KCl,MgSO₄), a buffering agent (a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), aprotease inhibitor, glycerol, and the like may be present in suchcompositions.

Pharmaceutical compositions are also provided. The pharmaceuticalcompositions include any of the bifunctional molecules of the presentdisclosure, and a pharmaceutically-acceptable carrier. Thepharmaceutical compositions generally include a therapeuticallyeffective amount of the bifunctional molecule. By “therapeuticallyeffective amount” is meant a dosage sufficient to produce a desiredresult, e.g., an amount sufficient to effect beneficial or desiredtherapeutic (including preventative) results, such as a reduction incellular proliferation in an individual having a cell proliferativedisorder (e.g., cancer) associated with the cell surface molecule orextracellular molecule to which the first moiety of the bifunctionalmolecule specifically binds, etc. An effective amount may beadministered in one or more administrations.

A bifunctional molecule of the present disclosure can be incorporatedinto a variety of formulations for therapeutic administration. Moreparticularly, the bifunctional molecule can be formulated intopharmaceutical compositions by combination with appropriatepharmaceutically acceptable excipients or diluents, and may beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, injections, inhalants and aerosols.

Formulations of the bifunctional molecules of the present disclosuresuitable for administration to an individual (e.g., suitable for humanadministration) are generally sterile and may further be free ofdetectable pyrogens or other contaminants contraindicated foradministration to an individual according to a selected route ofadministration.

In pharmaceutical dosage forms, the bifunctional molecule can beadministered alone or in appropriate association, as well as incombination, with other pharmaceutically-active compounds. The followingmethods and excipients are merely examples and are in no way limiting.

For oral preparations, the bifunctional molecule can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The bifunctional molecules can be formulated into preparations forinjection by dissolving, suspending or emulsifying them in an aqueous ornon-aqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

The pharmaceutical composition may be in a liquid form, a lyophilizedform or a liquid form reconstituted from a lyophilized form, where thelyophilized preparation is to be reconstituted with a sterile solutionprior to administration. The standard procedure for reconstituting alyophilized composition is to add back a volume of pure water (typicallyequivalent to the volume removed during lyophilization); howeversolutions comprising antibacterial agents may be used for the productionof pharmaceutical compositions for parenteral administration.

An aqueous formulation of the bifunctional molecule may be prepared in apH-buffered solution, e.g., at pH ranging from about 4.0 to about 8.0,such as from about 4.5 to about 7.5, e.g., from about 5.0 to about 7.0.Examples of buffers that are suitable for a pH within this range includephosphate-, histidine-, citrate-, succinate-, acetate-buffers and otherorganic acid buffers. The buffer concentration can be from about 1 mM toabout 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on thebuffer and the desired tonicity of the formulation.

Methods of Use

As summarized above, also provides are methods of using the bifunctionalmolecules of the present disclosure. In some embodiments, the methodsincluding using any of the bifunctional molecules described in theBifunctional Molecule section above, which is incorporated but notreiterated herein for purposes of brevity.

In certain aspects, provided are methods of degrading a cell surfacemolecule or extracellular molecule. Such methods include contacting thecell surface molecule or extracellular molecule with any of thebifunctional molecules of the present disclosure, under conditions inwhich the lysosomal targeting molecule shuttles the cell surfacemolecule or extracellular molecule to the lysosome for degradation. Suchmethods find use in a variety of applications. In certain aspects, themethod is performed in vitro (e.g., in a tube, cell culture plate orwell, or the like) and finds use, e.g., in testing and/or researchapplications. In other aspects, the method is performed in vivo (e.g.,in an individual to whom the bifunctional molecule is administered) andfinds use, e.g., in clinical/therapeutic applications.

In some embodiments, provided are methods that include administering toan individual in need thereof a therapeutically effective amount of anyof the bifunctional molecules or any of the pharmaceutical compositionsof the present disclosure. A variety of individuals are treatableaccording to the subject methods. Generally such subjects are “mammals”or “mammalian,” where these terms are used broadly to describe organismswhich are within the class mammalia, including the orders carnivore(e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), andprimates (e.g., humans, chimpanzees, and monkeys). In some embodiments,the individual is a human.

In some embodiments, an effective amount of the bifunctional molecule(or pharmaceutical composition including same) is an amount that, whenadministered alone (e.g., in monotherapy) or in combination (e.g., incombination therapy) with one or more additional therapeutic agents, inone or more doses, is effective to reduce the symptoms of a medicalcondition of the individual (e.g., cancer, neurodegenerative disorder,etc.) by at least about 5%, at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, or more, compared to the symptomsin the individual in the absence of treatment with the bifunctionalmolecule or pharmaceutical composition.

In certain aspects, the individual has, or is suspected of having, aneurodegenerative disorder characterized by amyloid-β deposition in thebrain (e.g., Alzheimer's Disease) or tau protein deposition in the brain(e.g., a tauopathy), and the first moiety of the bifunctional moleculespecifically binds to apoE4 (e.g., apoE4 expressed from the E4 allele ofthe APOE4 gene), such that targeted degradation of apoE4 treats theindividual's neurodegenerative disorder.

In some embodiments, provided are methods that include administering toan individual having cancer a therapeutically effective amount of any ofthe bifunctional molecules or any of the pharmaceutical compositions ofthe present disclosure. According to such methods, the first moiety ofthe bifunctional molecule specifically binds a cell surface molecule orextracellular molecule that at least contributes to individual's cancer,and where targeted degradation of the cell surface molecule orextracellular molecule using the bifunctional molecule treats theindividual's cancer. In certain aspects, the first moiety specificallybinds to a molecule selected from a cell surface molecule on a cancercell, a ligand for a cell surface molecule on a cancer cell, a cellsurface molecule on an immune cell, a ligand for a cell surface moleculeon an immune cell, an inhibitory immune receptor, and a ligand of aninhibitory immune receptor.

In certain embodiments, the individual has a cancer characterized by thepresence of a solid tumor, a semi-solid tumor, a primary tumor, ametastatic tumor, or the like. In some embodiments, the individual has acancer selected from breast cancer, melanoma, lung cancer, colorectalcancer, prostate cancer, glioma, bladder cancer, endometrial cancer,kidney cancer, leukemia (e.g., acute myeloid leukemia (AML)) livercancer (e.g., hepatocellular carcinoma (HCC), such as primary orrecurrent HCC), non-Hodgkin lymphoma, pancreatic cancer, thyroid cancer,any combinations thereof, and any sub-types thereof.

According to some embodiments, the individual has a particular liverdisease (including but not limited to hepatocellular carcinoma (HCC)),and the methods are for treating the disease. For example, in certainembodiments, the individual has HCC, the first moiety binds a cellsurface molecule on HCC cells of the individual, and the second moietybinds ASGPR. In certain embodiments, the first moiety binds atumor-promoting protein on HCC cells of the individual. According tosome embodiments, the tumor-promoting protein is a growth factor on theHCC cells. Non-limiting examples of such growth factors include EGFR,C-Met, IGF1R, and FGFR4. Such a bifunctional molecule may include any ofthe second moieties that bind ASGPR as described elsewhere herein.

In certain embodiments, the individual has fibrosis of the liver, andthe methods are for treating the liver fibrosis. For example, accordingto some embodiments, the individual has liver fibrosis, the first moietybinds a cell surface molecule on fibrotic liver cells of the individual,and the second moiety binds ASGPR. In certain embodiments, the firstmoiety binds a fibrosis-promoting protein on the fibrotic liver cells ofthe individual. According to some embodiments, the fibrosis-promotingprotein is a growth factor on the fibrotic liver cells. A non-limitingexample of such a growth factor is PDGFR. Such a bifunctional moleculemay include any of the second moieties that bind ASGPR as describedelsewhere herein.

In any of the methods of using the bifunctional molecules of the presentdisclosure, in certain embodiments, the bifunctional molecule enhancesdegradation of the cell surface molecule or extracellular moleculerelative to degradation of the cell surface molecule or extracellularmolecule in the presence of the first moiety alone. Similarly, in any ofthe methods of using the bifunctional molecules of the presentdisclosure, according to some embodiments, the bifunctional moleculeenhances degradation of the cell surface molecule or extracellularmolecule relative to degradation of the cell surface molecule orextracellular molecule in the presence of the first moiety or the secondmoiety alone. Details regarding such enhancement of degradation areprovided in the Bifunctional Molecules section above and incorporatedbut not reiterated herein for purposes of brevity.

By “treat”, “treating” or “treatment” is meant at least an ameliorationof the symptoms associated with the medical condition (e.g., cellproliferative disorder, e.g., cancer) of the individual, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with themedical condition being treated. As such, treatment also includessituations where the medical condition, or at least symptoms associatedtherewith, are completely inhibited, e.g., prevented from happening, orstopped, e.g., terminated, such that the individual no longer suffersfrom the medical condition, or at least the symptoms that characterizethe medical condition.

In certain aspects, the present disclosure provides methods of enhancingantibody-dependent cellular cytotoxicity (ADCC) including administeringto an individual in need of ADCC a bifunctional molecule orpharmaceutical composition of the present disclosure. In someembodiments, the first moiety of the bifunctional molecule specificallybinds to an inhibitory immune receptor or a ligand of an inhibitoryimmune receptor. In certain aspects, the first moiety of thebifunctional molecule specifically binds to an immune checkpointmolecule such as PD-1, PD-L1, CTLA4, TIM3, LAG3, TIGIT, or a member ofthe B7 family.

The bifunctional molecule or pharmaceutical composition may beadministered to the individual using any available method and routesuitable for drug delivery, including in vivo and ex vivo methods, aswell as systemic and localized routes of administration. Conventionaland pharmaceutically acceptable routes of administration includeintranasal, intramuscular, intra-tracheal, subcutaneous, intradermal,topical application, ocular, intravenous, intra-arterial, nasal, oral,and other enteral and parenteral routes of administration. In someembodiments, the administering is by parenteral administration. Routesof administration may be combined, if desired, or adjusted dependingupon the bifunctional molecule and/or the desired effect. Thebifunctional molecules or pharmaceutical compositions may beadministered in a single dose or in multiple doses. In some embodiments,the bifunctional molecule or pharmaceutical composition is administeredintravenously. In some embodiments, the bifunctional molecule orpharmaceutical composition is administered by injection, e.g., forsystemic delivery (e.g., intravenous infusion) or to a local site.

Kits

As summarized above, the present disclosure also provides kits. In someembodiments, a subject kit includes any of the bifunctional molecules ofthe present disclosure (including any of the bifunctional moleculesdescribed in the Bifunctional Molecule section above, which isincorporated but not reiterated herein for purposes of brevity), andinstructions for using the bifunctional molecule to degrade the cellsurface molecule or extracellular molecule to which the first moietyspecifically binds. In certain aspects, the instructions are fordegrading the cell surface molecule or extracellular molecule in vitro,e.g., for research and/or testing purposes. In other aspects, theinstructions are for degrading the cell surface molecule orextracellular molecule in vivo, e.g., for clinical/therapeuticapplications. For example, provided are kits that include any of thebifunctional molecules or pharmaceutical compositions of the presentdisclosure, and instructions for administering the bifunctional moleculeor pharmaceutical composition to an individual in need thereof. Suchkits may include a quantity of the bifunctional molecule orpharmaceutical composition, present in unit dosages, e.g., ampoules, ora multi-dosage format. As such, in certain embodiments, the kits mayinclude one or more (e.g., two or more) unit dosages (e.g., ampoules) ofthe bifunctional molecule or pharmaceutical composition.

The term “unit dosage”, as used herein, refers to physically discreteunits suitable as unitary dosages for human and animal subjects, eachunit containing a predetermined quantity of the composition calculatedin an amount sufficient to produce the desired effect. The amount of theunit dosage depends on various factors, such as the particularbifunctional molecule employed, the effect to be achieved, and thepharmacodynamics associated with the bifunctional molecule, in theindividual. In yet other embodiments, the kits may include a singlemulti dosage amount of the bifunctional molecule or pharmaceuticalcomposition.

In other aspects, provided are kits that include any of theglycopolymers of the present disclosure (including any of theglycopolymers described in the Bifunctional Molecule section above,which is incorporated but not reiterated herein for purposes ofbrevity), and instructions for conjugating the glycopolymer to amolecule of interest. Such kits may further include reagents forconjugating the glycopolymer to a molecule of interest. In someembodiments, the molecule of interest is a polypeptide. Non-limitingexamples of such polypeptides include antibodies. In certain aspects,the molecule of interest specifically binds a cell surface molecule orextracellular molecule, including any of the cell surface molecules orextracellular molecules described in the Bifunctional Molecule sectionabove, which is incorporated but not reiterated herein for purposes ofbrevity.

Components of the kits may be present in separate containers, ormultiple components may be present in a single container.

The instructions included in the kits may be recorded on a suitablerecording medium. For example, the instructions may be printed on asubstrate, such as paper or plastic, etc. As such, the instructions maybe present in the kits as a package insert, in the labeling of thecontainer of the kit or components thereof (i.e., associated with thepackaging or sub-packaging) etc. In other embodiments, the instructionsare present as an electronic storage data file present on a suitablecomputer readable storage medium, e.g., portable flash drive, DVD,CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, the means for obtaining theinstructions is recorded on a suitable substrate.

Glycopolymers, Monomers, and Methods of Making Same

The present disclosure also provides glycopolymers. In some embodiments,a glycopolymer of the present disclosure includes a polymer scaffold andone or more mannose-6-phosphate receptor (M6PR) ligands attached to thepolymer scaffold. The glycopolymer, polymer scaffold, and/or M6PRligands may be any of those described in the Bifunctional Moleculesection above, which is incorporated but not reiterated herein forpurposes of brevity. For example, the glycopolymer may be a glycoproteinincluding one or more amino acids functionalized with the one or moreM6PR ligands. The glycoprotein may be a N-carboxyanhydride (NCA)-derivedglycoprotein. In certain aspects, the one or more M6PR ligands includeone or more mannose-6-phosphates (M6P). Alternatively, or additionally,the one or more M6PR ligands include one or more M6P analogs, e.g., anyof the M6P analogs described herein, such as one or moremannose-6-phosphonates (M6Pn). In some embodiments, the polymer scaffoldincludes from 1 to 50 M6PR ligands, such as from 1 to 40, 1 to 30, 1 to20, 1 to 10 (e.g., 1 to 6), or 1 to 5 M6PR ligands. In certain aspects,the polymer scaffold includes from 10 to 50, 15 to 45, 20 to 40, or 25to 35 M6PR ligands. In certain aspects, the polymer scaffold includes 5or more, 10 or more, 20 or more, 30 or more, or 40 or more M6PR ligands.

Also provided are methods of making the glycopolymers of the presentdisclosure. In some embodiments, making the glycopolymer includespolymerization. The polymerization may be by NCA polymerization, anexample of which is schematically illustrated in FIG. 8.

In certain aspects, a method of making the glycopolymer includesattaching the one or more M6PR ligands to the polymer scaffold. In otheraspects, such methods include synthesizing the polymer scaffold frommonomers functionalized with the one or more M6PR ligands. For example,the scaffold may be synthesized from one or more monomers functionalizedwith the one or more M6PR ligands, where the synthesizing is bysolid-phase synthesis. An example solid-phase synthesis scheme isprovided in FIG. 9.

In related aspects, the present disclosure provides monomers. Themonomers are functionalized with one or more mannose-6-phosphatereceptor (M6PR) ligands. In certain aspects, the monomers are aminoacids. In some embodiments, the monomers are non-natural amino acids.The one or more M6PR ligands may include one or moremannose-6-phosphates (M6P). Alternatively, or additionally, the one ormore M6PR ligands may include one or more M6P analogs, e.g., any of theM6P analogs described herein, such as mannose-6-phosphonates (M6Pn).

Notwithstanding the appended claims, the present disclosure is alsodefined by the following embodiments.

1. A bifunctional molecule comprising:

-   -   a first moiety that specifically binds a cell surface molecule        or extracellular molecule; and    -   a second moiety that specifically binds a lysosomal targeting        molecule.        2. The bifunctional molecule of embodiment 1, wherein the        bifunctional molecule enhances degradation of the cell surface        molecule or extracellular molecule relative to degradation of        the cell surface molecule or extracellular molecule in the        presence of the first moiety alone.        3. The bifunctional molecule of embodiment 1 or embodiment 2,        wherein the first moiety specifically binds a cell surface        molecule.        4. The bifunctional molecule of embodiment 3, wherein the cell        surface molecule is a cell surface receptor.        5. The bifunctional molecule of embodiment 4, wherein the cell        surface receptor is a growth factor receptor.        6. The bifunctional molecule of any one of embodiments 1 to 5,        wherein the cell surface molecule is present on a cancer cell.        7. The bifunctional molecule of embodiment 6, wherein the cell        surface molecule is a tumor-associated antigen or a        tumor-specific antigen.        8. The bifunctional molecule of any one of embodiments 1 to 7,        wherein the cell surface molecule is present on an immune cell.        9. The bifunctional molecule of embodiment 8, wherein the immune        cell is selected from the group consisting of: a natural killer        (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic        cell, a T cell, a B cell, a mast cell, a basophil, and an        eosinophil.        10. The bifunctional molecule of embodiment 8, wherein the cell        surface molecule is an inhibitory immune receptor.        11. The bifunctional molecule of embodiment 10, wherein the cell        surface molecule is a ligand of an inhibitory immune receptor.        12. The bifunctional molecule of embodiment 8, wherein the cell        surface molecule is an immune checkpoint molecule.        13. The bifunctional molecule of embodiment 12, wherein the        immune checkpoint molecule is selected from the group consisting        of: PD-1, PD-L1, CTLA4, TIM3, LAG3, TIGIT, and a member of the        B7 family.        14. The bifunctional molecule of embodiment 1, wherein the first        moiety specifically binds an extracellular molecule.        15. The bifunctional molecule of embodiment 14, wherein the        extracellular molecule is a ligand for a cell surface receptor.        16. The bifunctional molecule of embodiment 15, wherein the        extracellular molecule is a growth factor.        17. The bifunctional molecule of embodiment 15, wherein the        extracellular molecule is a cytokine or a chemokine.        18. The bifunctional molecule of embodiment 14, wherein the        extracellular molecule is an antibody.        19. The bifunctional molecule of embodiment 18, wherein the        antibody is an autoantibody.        20. The bifunctional molecule of embodiment 18 or embodiment 19,        wherein the antibody specifically binds to a cell surface        molecule or an extracellular molecule.        21. The bifunctional molecule of any one of embodiments 1 to 20,        wherein the first moiety is selected from the group consisting        of: a polypeptide, a ligand, an aptamer, a nanoparticle, and a        small molecule.        22. The bifunctional molecule of embodiment 21, wherein the        first moiety is a polypeptide.        23. The bifunctional molecule of embodiment 22, wherein the        first moiety is an antibody.        24. The bifunctional molecule of embodiment 23, wherein the        antibody is an IgG, a single chain Fv (scFv), Fab, (Fab)₂,        (scFv′)₂, or a nanobody.        25. The bifunctional molecule of any one of embodiments 1 to 24,        wherein the second moiety is selected from the group consisting        of: a polypeptide, a ligand, an aptamer, a nanoparticle, and a        small molecule.        26. The bifunctional molecule of any one of embodiments 1 to 25,        wherein the lysosomal targeting molecule is a        mannose-6-phosphate receptor (M6PR).        27. The bifunctional molecule of embodiment 26, wherein the        second moiety comprises one or more M6PR ligands.        28. The bifunctional molecule of embodiment 27, wherein the one        or more M6PR ligands comprise one or more mannose-6-phosphates        (M6P).        29. The bifunctional molecule of embodiment 27 or embodiment 28,        wherein the one or more M6PR ligands comprise one or more M6P        analogs.        30. The bifunctional molecule of embodiment 29, wherein the one        or more M6P analogs comprise one or more mannose-6-phosphonates        (M6Pn).        31. The bifunctional molecule of any one of embodiments 27 to        30, wherein the second moiety comprises from 1 to 500 M6PR        ligands.        32. The bifunctional molecule of any one of embodiments 27 to        31, wherein the second moiety comprises a polymer scaffold that        displays the one or more M6PR ligands.        33. The bifunctional molecule of embodiment 32, wherein the        polymer scaffold is a glycopolymer comprising the one or more        M6PR ligands.        34. The bifunctional molecule of embodiment 33, wherein the        glycopolymer is a glycoprotein comprising one or more amino        acids functionalized with the one or more M6PR ligands.        35. The bifunctional molecule of embodiment 34, wherein the        glycoprotein is a N-carboxyanhydride (NCA)-derived glycoprotein.        36. The bifunctional molecule of any one of embodiments 1 to 25,        wherein the lysosomal targeting molecule is expressed on the        surface of liver cells.        37. The bifunctional molecule of embodiment 36, wherein the        lysosomal targeting molecule is expressed on the surface of        hepatocytes.        38. The bifunctional molecule of embodiment 36 or embodiment 37,        wherein the lysosomal targeting molecule is expressed on the        surface of hepatocellular carcinoma (HCC) cells, fibrotic liver        cells, or both.        39. The bifunctional molecule of any one of embodiments 36 to        38, wherein the lysosomal targeting molecule is        asialoglycoprotein receptor (ASGPR).        40. The bifunctional molecule of embodiment 39, wherein the        second moiety comprises one or more ASGPR ligands.        41. The bifunctional molecule of embodiment 40, wherein the one        or more ASGPR ligands comprises one or more        N-acetylgalactosamines (GalNAc).        42. The bifunctional molecule of embodiment 40 or embodiment 41,        wherein the one or more ASGPR ligands comprises one or more        galactoses.        43. The bifunctional molecule of any one of embodiments 40 to        42, wherein the one or more ASGPR ligands comprises one or more        glucoses.        44. The bifunctional molecule of any one of embodiments 40 to        43, wherein the second moiety comprises from 1 to 500 ASGPR        ligands.        45. The bifunctional molecule of any one of embodiments 40 to        44, wherein the second moiety comprises a polymer comprising the        one or more ASGPR ligands.        46. The bifunctional molecule of embodiment 45, wherein the        second moiety comprises poly(GalNAc-co-Ala).        47. The bifunctional molecule of embodiment 41, wherein the        second moiety comprises a monovalent, bivalent, or trivalent        GalNAc-containing dendrimer scaffold.        48. The bifunctional molecule of embodiment 47, wherein the        second moiety comprises a trivalent GalNAc-containing dendrimer        scaffold.        49. The bifunctional molecule of embodiment 42, wherein the        second moiety comprises a monovalent, bivalent, or trivalent        galactose-containing dendrimer scaffold.        50. The bifunctional molecule of embodiment 49, wherein the        second moiety comprises a trivalent galactose-containing        dendrimer scaffold.        51. The bifunctional molecule of any one of embodiments 36 to        50, wherein the first moiety specifically binds a cell surface        molecule expressed on hepatocytes.        52. The bifunctional molecule of embodiment 51, wherein the cell        surface molecule is a growth factor receptor.        53. The bifunctional molecule of embodiment 52, wherein the        growth factor receptor is selected from the group consisting of        epidermal growth factor receptor (EGFR), C-Met, insulin like        growth factor 1 receptor (IGF1R), fibroblast growth factor        receptor 4 (FGFR4), and platelet-derived growth factor receptor        (PDGFR).        54. The bifunctional molecule of any one of embodiments 1 to 53,        wherein the first moiety is a polypeptide and the second moiety        is a polypeptide, and wherein the bifunctional molecule is a        fusion protein comprising the first moiety fused to the second        moiety.        55. The bifunctional molecule of embodiment 54, wherein the        first moiety is fused directly to the second moiety.        56. The bifunctional molecule of embodiment 54, comprising a        spacer domain between the first moiety and the second moiety.        57. The bifunctional molecule of any one of embodiments 1 to 56,        wherein the bifunctional molecule is a bispecific antibody that        specifically binds:    -   a cell surface molecule or extracellular molecule; and    -   a lysosomal targeting molecule.        58. The bifunctional molecule of any one of embodiments 1 to 53,        wherein the bifunctional molecule is a conjugate comprising the        first moiety conjugated to the second moiety.        59. The bifunctional molecule of embodiment 58, wherein the        first moiety is an antibody.        60. The bifunctional molecule of embodiment 58 or embodiment 59,        comprising a second moiety as defined in any one of embodiments        27 to 35.        61. The bifunctional molecule of embodiment 58 or embodiment 59,        comprising a second moiety as defined in any one of embodiments        40 to 50.        62. A nucleic acid encoding the bifunctional molecule of any one        of embodiments 54 to 57.        63. An expression vector comprising the nucleic acid of        embodiment 62.        64. A cell comprising the nucleic acid of embodiment 62 or the        expression vector of embodiment 63.        65. A method of producing the cell of embodiment 64, comprising        introducing into a cell the nucleic acid of embodiment 62 or the        expression vector of embodiment 63.        66. A method of making the bifunctional molecule of embodiment        58 or embodiment 59, comprising conjugating the first moiety to        the second moiety.        67. The method according to embodiment 66, wherein the        conjugating comprises site-specifically conjugating the first        moiety to the second moiety.        68. The method according to embodiment 67, wherein the first        moiety comprises a polypeptide, and wherein the conjugating        comprises site-specifically conjugating the second moiety to a        pre-selected amino acid of the first moiety.        69. The method according to embodiment 68, wherein the        pre-selected amino acid is at the N-terminus or C-terminus of        the first moiety.        70. The method according to embodiment 68, wherein the        pre-selected amino acid is internal to the first moiety.        71. The method according to any one of embodiments 68 to 70,        wherein the pre-selected amino acid is a non-natural amino acid.        72. The method according to any one of embodiments 66 to 71,        wherein the first moiety is an antibody.        73. The method according to any one of embodiments 66 to 72,        wherein the second moiety is as defined in any one of        embodiments 27 to 35.        74. The method according to any one of embodiments 66 to 72,        wherein the second moiety is as defined in any one of        embodiments 40 to 50.        75. The method according to any one of embodiments 66 to 74,        wherein the conjugating is by alkyne-azide cycloaddition.        76. A method of degrading a cell surface molecule or        extracellular molecule, comprising:    -   contacting the cell surface molecule or extracellular molecule        with the bifunctional molecule of any one of embodiments 1 to 61        under conditions in which the lysosomal targeting molecule        shuttles the cell surface molecule or extracellular molecule to        the lysosome for degradation.        77. The method according to embodiment 76, wherein the        bifunctional molecule enhances degradation of the cell surface        molecule or extracellular molecule relative to degradation of        the cell surface molecule or extracellular molecule in the        presence of the first moiety alone.        78. The method according to embodiment 76 or embodiment 77,        wherein the method is performed in vitro.        79. The method according to embodiment 76 or embodiment 77,        wherein the method is performed in vivo.        80. A pharmaceutical composition comprising:    -   the bifunctional molecule of any one of embodiments 1 to 61; and    -   a pharmaceutically acceptable carrier.        81. The pharmaceutical composition of embodiment 80, wherein the        composition is formulated for parenteral administration.        82. A method comprising administering to an individual in need        thereof the pharmaceutical composition of embodiment 80 or        embodiment 81.        83. A method of treating cancer comprising administering to an        individual having cancer an effective amount of the        pharmaceutical composition of embodiment 80 or embodiment 81.        84. The method according to embodiment 83, wherein the first        moiety specifically binds to a molecule selected from the group        consisting of: a cell surface molecule on a cancer cell, a        ligand for a cell surface molecule on a cancer cell, a cell        surface molecule on an immune cell, a ligand for a cell surface        molecule on an immune cell, an inhibitory immune receptor, and a        ligand of an inhibitory immune receptor.        85. The method according to embodiment 83 or embodiment 84,        wherein the individual has hepatocellular carcinoma (HCC), the        first moiety binds a cell surface molecule on HCC cells of the        individual, and the second moiety binds ASGPR.        86. The method according to embodiment 85, wherein the first        moiety binds a growth factor on HCC cells of the individual.        87. The method according to embodiment 90, wherein the first        moiety binds a growth factor selected from the group consisting        of: EGFR, C-Met, IGF1R, and FGFR4.        88. The method according to any one of embodiments 85 to 87,        wherein the second moiety is as defined in any one of        embodiments 36 to 50.        89. A method of enhancing antibody-dependent cellular        cytotoxicity (ADCC) comprising administering to an individual in        need of ADCC the pharmaceutical composition of embodiment 80 or        embodiment 81.        90. A method of enhancing immunogenicity of a cancer in an        individual, comprising administering to the individual the        pharmaceutical composition of embodiment 80 or embodiment 81.        91. The method according to embodiment 89 or embodiment 90,        wherein the first moiety specifically binds to a molecule        selected from the group consisting of: an inhibitory immune        receptor, and a ligand of an inhibitory immune receptor.        92. The method according to any one of embodiments 82 to 91,        wherein the administering is by parenteral administration.        93. The method according to any one of embodiments 82 to 91,        wherein the bifunctional molecule enhances degradation of the        cell surface molecule or extracellular molecule relative to        degradation of the cell surface molecule or extracellular        molecule in the presence of the first moiety alone.        94. A kit comprising:    -   the bifunctional molecule of any one of embodiments 1 to 61; and    -   instructions for degrading the cell surface molecule or        extracellular molecule to which the first moiety specifically        binds.        95. The kit of embodiment 94, wherein the instructions are for        degrading the cell surface molecule or extracellular molecule in        vitro.        96. The kit of embodiment 94, wherein the instructions are for        degrading the cell surface molecule or extracellular molecule in        vivo.        97. A kit comprising:    -   the bifunctional molecule of any one of embodiments 1 to 61 or        the pharmaceutical composition of embodiment 80 or embodiment        81; and    -   instructions for administering the bifunctional molecule or        pharmaceutical composition to an individual in need thereof.        98. The kit of embodiment 97, wherein the bifunctional molecule        or pharmaceutical composition is present in one or more unit        dosages.        99. The kit of embodiment 97, wherein the bifunctional molecule        or pharmaceutical composition is present in two or more unit        dosages.        100. A glycopolymer comprising:    -   a polymer scaffold; and    -   one or more mannose-6-phosphate receptor (M6PR) ligands attached        to the polymer scaffold.        101. The glycopolymer of embodiment 100, wherein the        glycopolymer is a glycoprotein comprising one or more amino        acids functionalized with the one or more M6PR ligands.        102. The glycopolymer of embodiment 101, wherein the        glycoprotein is a N-carboxyanhydride (NCA)-derived glycoprotein.        103. The glycopolymer of any one of embodiments 100 to 102,        wherein the one or more M6PR ligands comprise one or more        mannose-6-phosphates (M6P).        104. The glycopolymer of any one of embodiments 100 to 103,        wherein the one or more M6PR ligands comprise one or more M6P        analogs.        105. The glycopolymer of embodiment 104, wherein the one or more        M6P analogs comprise one or more mannose-6-phosphonates (M6Pn).        106. The glycopolymer of any one of embodiments 100 to 105,        wherein the polymer scaffold comprises from 1 to 500 M6PR        ligands.        107. A method of making the glycopolymer of any one of        embodiments 100 to 106, comprising:    -   attaching the one or more M6PR ligands to the polymer scaffold;        or    -   synthesizing the polymer scaffold from monomers functionalized        with the one or more M6PR ligands.        108. The method according to embodiment 107, wherein the        scaffold is polymerized from one or more monomers functionalized        with the one or more M6PR ligands, and wherein the synthesizing        is by solid-phase synthesis.        109. The method according to embodiment 108, wherein the        glycopolymer is a glycoprotein polymer, and wherein the        synthesizing is by solid-phase peptide synthesis.        110. A kit comprising:    -   the glycopolymer of any one of embodiments 100 to 106; and    -   instructions for conjugating the glycopolymer to a molecule of        interest.        111. The kit of embodiment 110, further comprising reagents for        conjugating the glycopolymer to a molecule of interest.        112. The kit of embodiment 110 or embodiment 111, wherein the        molecule of interest is a polypeptide.        113. The kit of embodiment 112, wherein the polypeptide is an        antibody.        114. The kit of any one of embodiments 110 to 113, wherein the        molecule of interest specifically binds a cell surface molecule        or extracellular molecule.        115. A monomer functionalized with one or more        mannose-6-phosphate receptor (M6PR) ligands.        116. The monomer of embodiment 115, wherein the monomer is an        amino acid.        117. The monomer of embodiment 115, wherein the monomer is a        non-natural amino acid.        118. The monomer of any one of embodiments 115 to 117, wherein        the one or more M6PR ligands comprise one or more        mannose-6-phosphates (M6P).        119. The monomer of any one of embodiments 115 to 118, wherein        the one or more M6PR ligands comprise one or more M6P analogs.        120. The monomer of embodiment 119, wherein the one or more M6P        analogs comprise one or more mannose-6-phosphonates (M6Pn).

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1—Mannose-6-Phosphate Polymers Shuttle Cargo toLysosomes

Tested in this example was a bifunctional molecule (see FIG. 10) thatincludes a biotin cap (the “first moiety” as used herein—depicted as atriangle in FIG. 10) and an M6Pn polymer (the “second moiety” as usedherein) to determine whether the bifunctional molecule could mediatetransfer of NeutrAvidin-647 (NA647—a protein to which biotin stronglybinds) to lysosomes from the extracellular space for degradation. FIG.10 provides fluorescence imaging results (bottom) demonstrating that thebifunctional molecule can indeed mediate transfer of NeutrAvidin-647 tolysosomes, as colocalization of both protein and lysosome staining dyeare observed.

Next, various cell lines were tested in a manner as described above.Shown in FIG. 11 is data demonstrating that several cell lines exhibituptake of NA647 in a M6Pn polymer-dependent manner. In view of theseresults, it is expected that any cell line bearing M6PRs (e.g., CIM6PRs)will allow for shuttling of cell surface and extracellular molecules tothe lysosome by this method, and is not limited to the cell lines testedin the present study.

Example 2—M6Pn-Conjugated Antibodies Shuttle Targets to Lysosomes

Tested in this example were bifunctional molecules in which the firstmoiety is an antibody that binds to a particular target of interest andthe second moiety is a M6Pn-containing glycoprotein.

Provided in FIG. 13 is a schematic illustration (top) and fluorescenceimaging data (bottom) demonstrating that poly(M6Pn) labeled antibodiescan shuttle their binding partners to lysosomes. In this example, amouse IgG-488 was incubated with an anti-mouse IgG antibody bearing thepoly(M6Pn) tag, with colocalization of both protein and lysosomestaining dye (merge).

Provided in FIG. 14 is a schematic illustration (top) and further data(bottom) demonstrating that poly(M6Pn) labeled antibodies can shuttletheir binding partners to intracellular compartments. In this example,recombinant human apoE4 was incubated with a mouse-derived anti-humanapoE4 antibody, an anti-mouse IgG antibody, or an anti-mouse antibodybearing the poly(M6Pn) tag. Significantly more uptake is observed withthe M6Pn-containing secondary antibody.

Provided in FIG. 15 is further data demonstrating that poly(M6Pn)labeled antibodies can shuttle their binding partners for degradation.In this example, EGFR degradation was assessed by incubating cells withcetuximab bearing an M6Pn tag. Loss of total EGFR is observed for allcell lines tested, compared to cetuximab or cetuximab bearing a mockpolymer (GalNAc). EGF is a positive control for EGFR degradation. Lane1: control. Lane 2: EGF (100 ng/mL, 1 h, +control). Lane 3: cetuximab.Lane 4: cetuximab-GalNAc conjugate. Lane 5: cetuximab-M6P conjugate(long). Lane 6: cetuximab-M6P conjugate (short). Percent of control wascalculated by densitometry.

Provided in FIG. 16 is data demonstrating that poly(M6Pn) labeledantibody fragments can shuttle their binding partners for degradation.In this example, EGFR degradation was assessed by incubating cells withcetuximab-derived Fab portions bearing an M6Pn tag. Loss of total EGFRis observed compared to cetuximab Fab alone or cetuximab Fab bearing amock polymer (GalNAc).

Provided in FIG. 17 is further data demonstrating that poly(M6Pn)labeled antibodies can shuttle their binding partners for degradation.In this example, degradation of CD71 (transferrin receptor) was assessedby incubating cells with a primary mouse-derived antibody against CD71,an anti-mouse IgG antibody, or an anti-mouse antibody bearing thepoly(M6Pn) tag. The system containing the M6P tag leads to significantlymore degradation.

Provided in FIG. 18 is further data demonstrating that poly(M6Pn)labeled antibodies can shuttle their binding partners for degradation.In this example, PDL1 degradation was assessed by incubating cells withanti-PDL1 antibody or anti-PDL1 antibody bearing an M6P tag. Degradationis only observed with M6P-labeled anti-PDL1 antibody.

Example 3—ASGPR Ligands Shuttle Cargo to Lysosomes in Hepatocytes

Several current therapies suffer from off target effects. Described inthis example is the targeted degradation of proteins in the liver whichmay be employed to treat liver-related diseases such as liver cancer andliver fibrosis. A scavenger receptor called asialoglycoprotein receptor(ASGPR) is exclusively or near-exclusively expressed in liver cells(hepatocytes). As schematically illustrated in FIG. 20 (left), ASGPRconstitutively recycles between the plasma membrane and the endosome.ASGPR brings extracellular glycoproteins inside the cell for degradationin the lysosome. Demonstrated herein is the harnessing of this receptorfor degrading extracellular or membrane proteins on hepatocytes using abifunctional molecule comprising a first moiety that binds to such amembrane or extracellular protein and a second moiety comprising ASGPRligands.

Tested in the ASGPR-related examples herein were bifunctional moleculesthat comprise an antibody that binds to a target molecule to be degradedconjugated to a second moiety comprising a polymer ofN-acetylgalactosamines (GalNAc), in particular a poly(GalNAc-co-Ala)polymer as shown in FIG. 20 (lower left).

To assess whether a bifunctional molecule comprising ASGPR ligands couldindeed shuttle cargo to lysosomes in hepatocytes, an assay schematicallyillustrated in FIG. 21 was employed. A bifunctional molecule comprisingan anti-mouse IgG antibody conjugated to a poly(GalNAc-co-Ala) polymerwas tested to determine whether the bifunctional molecule could shuttleextracellular fluorescent-labeled mouse IgG (IgG-AF647) into theintracellular compartment of hepatocytes. Also tested in this examplewas a bifunctional molecule comprising the anti-mouse IgG antibodyconjugated to M6PR ligands. HEPG2 cells (hepatocellular carcinoma cellline) were incubated with 50 nM IgG-AF647 and 25 nM of anti-mouseconjugates for 1 hour. Cellular uptake was analyzed by flow cytometry.As shown by the bar graph, an approximately 8-fold increase in cellfluorescence was observed using the GalNAc-containing conjugate whilethe M6Pn-containing conjugate induced 2-fold increase over background.The data demonstrate that in hepatocytes, where the expression level ofASGPR is higher than that of M6PR, the GalNAc-containing conjugate ismore effective in triggering cellular uptake.

Shown in FIG. 22 are results from the assay described for FIG. 21 but inHUH7 cells (another hepatocellular carcinoma cell line). Efficientuptake of IgG-AF647 into the HUH7 cells was observed. In addition,controls were included in which the cells were incubated with M6PR orASGPR inhibitors (monomeric M6P (mM6P) and monomeric GalNAc (mGalNAc),respectively). Uptake was reduced when the cells were incubated with theinhibitors, indicating that the uptake with GalNAc- or M6Pn-containingconjugates is indeed mediated by ASGPR or M6PR, respectively.

Example 4—Efficient Degradation of EGFR in Hepatocytes UsingCetuximab-ASGPR Ligand Conjugates

Epidermal growth factor receptor (EGFR) is known to induce proliferationand angiogenesis in hepatocellular carcinoma (HCC). 68% of HCC patientsexpress EGFR on their HCC cells. Transplantation currently remains thebest treatment option for patients with HCC, and the supply ofgood-quality deceased donor organs is limited. Receptor-tyrosine kinase(RTK) inhibitors or antibodies are often used for treatment, but HCCcells develop resistance to these treatments due to theheterodimerization of the receptor tyrosine kinases (EGFR, HER2, HER3,c-Met, IGF1R) which leads to the phosphorylation of the same downstreameffectors to restore oncogenic signaling via RTK crosstalk. Cetuximab,an EGFR blocking antibody, failed in Phase II Clinical Trials in HCCpatients.

Assessed in this example was whether a bifunctional molecule comprisingan anti-EGFR antibody (Cetuximab in this example) conjugated to ASGPRligands (poly(GalNAc-co-Ala) in this example) could induce uptake anddegradation of EGFR in HCC cells, as schematically illustrated in FIG.23 (top). Also tested were Cetuximab-M6PR ligand conjugates.

HEP3B cells were used in a first experiment. The cells were incubatedwith 10 nM of cetuximab conjugates for 48 hours and then lysed forwestern blot analysis. The western blot (shown in FIG. 23, bottom) has 5lanes: 1) no treatment, 2) EGF (known down-regulator of EGFR), 3)cetuximab, 4) cetuximab-GalNAc conjugate, and 5) cetuximab-M6Pnconjugate. As shown, the Cetuximab-GalNAc conjugate exhibited efficientEGRFR degradation and greater EGFR degradation as compared to theCetuximab-M6Pn conjugate. Vinculin was used as a loading control.

HEPG2 cells were used in a second experiment. Results are shown in FIG.24. Efficient degradation of EGFR in HEPG2 cells was observed. Here, thedegradation efficiency was similar between the cetuximab-GalNAc andcetuximab-M6Pn conjugates, possibly due to the relative levels of EGFRin the HEP3B and HEPG2 cell lines. Shown on the right are relative mRNAlevels of the receptors and EGFR in the two cell lines (available inpublic database). Since EGFR levels are relatively low in HEPG2 cellscompared to HEP3B cells, the data suggest that even with a lesseffective degrader (cetuximab-M6Pn conjugate), most of the membrane EGFRis being degraded, and that residual EGFR seen in the western blot maybe internal EGFR. ASGPR levels were also monitored to show that thelevel of ASGPR remained constant throughout the different treatments.

Next, a time-course study was performed to assess EGFR degradation overtime in HEP3B cells treated with the cetuximab conjugates. Results areshown in FIG. 25. As shown, by 12 hours, the cetuximab-GalNAc conjugatesreduces EGFR levels below 50%. The degradation increases at 48 hours.The cetuximab-M6Pn conjugate did not reduce EGFR levels below 50% at anyof the time points tested.

Immunofluorescence experiments were performed to assess whether theresidual EGFR is membrane EGFR or intracellular EGFR. Results are shownin FIG. 26. The outlines of the cells in HEP3B and cetuximab indicateEGFR localization on the membrane. When HEP3B was treated withcetuximab-M6Pn conjugate, some of the EGFR is localized internally whilesome remains on the membrane. When HEP3B is treated withcetuximab-GalNAc conjugate, almost no EGFR is observed on the membrane,and the vast majority of the EGFR is inside the cells. Accordingly, thecetuximab-GalNAc conjugate degraded most membrane-bound EGFR, and theresidual (approx. 30%) EGFR seen on the western blot appears to beintracellular EGFR.

Example 5—HER2 Degradation Via Trastuzumab Alone and TrastuzumabConjugates

Assessed in this example was the extent of degradation of HER2 in HUH7and HEPG2 cells in the presence of Trastuzumab alone or Trastuzumabconjugated to the GalNAc-containing polymer shown in FIG. 20(“Trastuzumab-GalNAc”). HUH7 and HEPG2 cells were incubated with 10 nMof trastuzumab or trastuzumab-GalNAc conjugates for 48 hours and thenlysed for western blot analysis. The western blot in FIG. 27 has 3 lanesfor each cell line: 1) no treatment, 2) trastuzumab, 3)trastuzumab-GalNAc. The bar graph in FIG. 27 shows average percent ofHER2 relative to the control in each cell line. As shown, there was nostatistical difference in HER2 degradation in the presence oftrastuzumab alone or trastuzumab conjugated to the GalNAc-containingpolymer in either cell line. As such, the Trastuzumab-GalNAc conjugatedid not enhance degradation of HER2 relative to degradation of HER2 inthe presence of Trastuzumab alone.

Accordingly, the preceding merely illustrates the principles of thepresent disclosure. It will be appreciated that those skilled in the artwill be able to devise various arrangements which, although notexplicitly described or shown herein, embody the principles of theinvention and are included within its spirit and scope. Furthermore, allexamples and conditional language recited herein are principallyintended to aid the reader in understanding the principles of theinvention and the concepts contributed by the inventors to furtheringthe art, and are to be construed as being without limitation to suchspecifically recited examples and conditions. Moreover, all statementsherein reciting principles, aspects, and embodiments of the invention aswell as specific examples thereof, are intended to encompass bothstructural and functional equivalents thereof. Additionally, it isintended that such equivalents include both currently known equivalentsand equivalents developed in the future, i.e., any elements developedthat perform the same function, regardless of structure. The scope ofthe present invention, therefore, is not intended to be limited to theexemplary embodiments shown and described herein.

What is claimed is:
 1. A bifunctional molecule comprising: a firstmoiety that specifically binds a cell surface molecule or extracellularmolecule; and a second moiety that specifically binds a lysosomaltargeting molecule.
 2. The bifunctional molecule of claim 1, wherein thebifunctional molecule enhances degradation of the cell surface moleculeor extracellular molecule relative to degradation of the cell surfacemolecule or extracellular molecule in the presence of the first moietyalone.
 3. The bifunctional molecule of claim 1 or claim 2, wherein thefirst moiety specifically binds a cell surface molecule.
 4. Thebifunctional molecule of claim 3, wherein the cell surface molecule is acell surface receptor.
 5. The bifunctional molecule of claim 4, whereinthe cell surface receptor is a growth factor receptor.
 6. Thebifunctional molecule of any one of claims 1 to 5, wherein the cellsurface molecule is present on a cancer cell.
 7. The bifunctionalmolecule of claim 6, wherein the cell surface molecule is atumor-associated antigen or a tumor-specific antigen.
 8. Thebifunctional molecule of any one of claims 1 to 7, wherein the cellsurface molecule is present on an immune cell.
 9. The bifunctionalmolecule of claim 8, wherein the immune cell is selected from the groupconsisting of: a natural killer (NK) cell, a macrophage, a monocyte, aneutrophil, a dendritic cell, a T cell, a B cell, a mast cell, abasophil, and an eosinophil.
 10. The bifunctional molecule of claim 8,wherein the cell surface molecule is an inhibitory immune receptor. 11.The bifunctional molecule of claim 10, wherein the cell surface moleculeis a ligand of an inhibitory immune receptor.
 12. The bifunctionalmolecule of claim 8, wherein the cell surface molecule is an immunecheckpoint molecule.
 13. The bifunctional molecule of claim 12, whereinthe immune checkpoint molecule is selected from the group consisting of:PD-1, PD-L1, CTLA4, TIM3, LAG3, TIGIT, and a member of the B7 family.14. The bifunctional molecule of claim 1, wherein the first moietyspecifically binds an extracellular molecule.
 15. The bifunctionalmolecule of claim 14, wherein the extracellular molecule is a ligand fora cell surface receptor.
 16. The bifunctional molecule of claim 15,wherein the extracellular molecule is a growth factor.
 17. Thebifunctional molecule of claim 15, wherein the extracellular molecule isa cytokine or a chemokine.
 18. The bifunctional molecule of claim 14,wherein the extracellular molecule is an antibody.
 19. The bifunctionalmolecule of claim 18, wherein the antibody is an autoantibody.
 20. Thebifunctional molecule of claim 18 or claim 19, wherein the antibodyspecifically binds to a cell surface molecule or an extracellularmolecule.
 21. The bifunctional molecule of any one of claims 1 to 20,wherein the first moiety is selected from the group consisting of: apolypeptide, a ligand, an aptamer, a nanoparticle, and a small molecule.22. The bifunctional molecule of claim 21, wherein the first moiety is apolypeptide.
 23. The bifunctional molecule of claim 22, wherein thefirst moiety is an antibody.
 24. The bifunctional molecule of claim 23,wherein the antibody is an IgG, a single chain Fv (scFv), Fab, (Fab)₂,(scFv′)₂, or a nanobody.
 25. The bifunctional molecule of any one ofclaims 1 to 24, wherein the second moiety is selected from the groupconsisting of: a polypeptide, a ligand, an aptamer, a nanoparticle, anda small molecule.
 26. The bifunctional molecule of any one of claims 1to 25, wherein the lysosomal targeting molecule is a mannose-6-phosphatereceptor (M6PR).
 27. The bifunctional molecule of claim 26, wherein thesecond moiety comprises one or more M6PR ligands.
 28. The bifunctionalmolecule of claim 27, wherein the one or more M6PR ligands comprise oneor more mannose-6-phosphates (M6P).
 29. The bifunctional molecule ofclaim 27 or claim 28, wherein the one or more M6PR ligands comprise oneor more M6P analogs.
 30. The bifunctional molecule of claim 29, whereinthe one or more M6P analogs comprise one or more mannose-6-phosphonates(M6Pn).
 31. The bifunctional molecule of any one of claims 27 to 30,wherein the second moiety comprises from 1 to 500 M6PR ligands.
 32. Thebifunctional molecule of any one of claims 27 to 31, wherein the secondmoiety comprises a polymer scaffold that displays the one or more M6PRligands.
 33. The bifunctional molecule of claim 32, wherein the polymerscaffold is a glycopolymer comprising the one or more M6PR ligands. 34.The bifunctional molecule of claim 33, wherein the glycopolymer is aglycoprotein comprising one or more amino acids functionalized with theone or more M6PR ligands.
 35. The bifunctional molecule of claim 34,wherein the glycoprotein is a N-carboxyanhydride (NCA)-derivedglycoprotein.
 36. The bifunctional molecule of any one of claims 1 to25, wherein the lysosomal targeting molecule is expressed on the surfaceof liver cells.
 37. The bifunctional molecule of claim 36, wherein thelysosomal targeting molecule is expressed on the surface of hepatocytes.38. The bifunctional molecule of claim 36 or claim 37, wherein thelysosomal targeting molecule is expressed on the surface ofhepatocellular carcinoma (HCC) cells, fibrotic liver cells, or both. 39.The bifunctional molecule of any one of claims 36 to 38, wherein thelysosomal targeting molecule is asialoglycoprotein receptor (ASGPR). 40.The bifunctional molecule of claim 39, wherein the second moietycomprises one or more ASGPR ligands.
 41. The bifunctional molecule ofclaim 40, wherein the one or more ASGPR ligands comprises one or moreN-acetylgalactosamines (GalNAc).
 42. The bifunctional molecule of claim40 or claim 41, wherein the one or more ASGPR ligands comprises one ormore galactoses.
 43. The bifunctional molecule of any one of claims 40to 42, wherein the one or more ASGPR ligands comprises one or moreglucoses.
 44. The bifunctional molecule of any one of claims 40 to 43,wherein the second moiety comprises from 1 to 500 ASGPR ligands.
 45. Thebifunctional molecule of any one of claims 40 to 44, wherein the secondmoiety comprises a polymer comprising the one or more ASGPR ligands. 46.The bifunctional molecule of claim 45, wherein the second moietycomprises poly(GalNAc-co-Ala).
 47. The bifunctional molecule of claim41, wherein the second moiety comprises a monovalent, bivalent, ortrivalent GalNAc-containing dendrimer scaffold.
 48. The bifunctionalmolecule of claim 47, wherein the second moiety comprises a trivalentGalNAc-containing dendrimer scaffold.
 49. The bifunctional molecule ofclaim 42, wherein the second moiety comprises a monovalent, bivalent, ortrivalent galactose-containing dendrimer scaffold.
 50. The bifunctionalmolecule of claim 49, wherein the second moiety comprises a trivalentgalactose-containing dendrimer scaffold.
 51. The bifunctional moleculeof any one of claims 36 to 50, wherein the first moiety specificallybinds a cell surface molecule expressed on hepatocytes.
 52. Thebifunctional molecule of claim 51, wherein the cell surface molecule isa growth factor receptor.
 53. The bifunctional molecule of claim 52,wherein the growth factor receptor is selected from the group consistingof epidermal growth factor receptor (EGFR), C-Met, insulin like growthfactor 1 receptor (IGF1R), fibroblast growth factor receptor 4 (FGFR4),and platelet-derived growth factor receptor (PDGFR).
 54. Thebifunctional molecule of any one of claims 1 to 53, wherein the firstmoiety is a polypeptide and the second moiety is a polypeptide, andwherein the bifunctional molecule is a fusion protein comprising thefirst moiety fused to the second moiety.
 55. The bifunctional moleculeof claim 54, wherein the first moiety is fused directly to the secondmoiety.
 56. The bifunctional molecule of claim 54, comprising a spacerdomain between the first moiety and the second moiety.
 57. Thebifunctional molecule of any one of claims 1 to 56, wherein thebifunctional molecule is a bispecific antibody that specifically binds:a cell surface molecule or extracellular molecule; and a lysosomaltargeting molecule.
 58. The bifunctional molecule of any one of claims 1to 53, wherein the bifunctional molecule is a conjugate comprising thefirst moiety conjugated to the second moiety.
 59. The bifunctionalmolecule of claim 58, wherein the first moiety is an antibody.
 60. Thebifunctional molecule of claim 58 or claim 59, comprising a secondmoiety as defined in any one of claims 27 to
 35. 61. The bifunctionalmolecule of claim 58 or claim 59, comprising a second moiety as definedin any one of claims 40 to
 50. 62. A nucleic acid encoding thebifunctional molecule of any one of claims 54 to
 57. 63. An expressionvector comprising the nucleic acid of claim
 62. 64. A cell comprisingthe nucleic acid of claim 62 or the expression vector of claim
 63. 65. Amethod of producing the cell of claim 64, comprising introducing into acell the nucleic acid of claim 62 or the expression vector of claim 63.66. A method of making the bifunctional molecule of claim 58 or claim59, comprising conjugating the first moiety to the second moiety. 67.The method according to claim 66, wherein the conjugating comprisessite-specifically conjugating the first moiety to the second moiety. 68.The method according to claim 67, wherein the first moiety comprises apolypeptide, and wherein the conjugating comprises site-specificallyconjugating the second moiety to a pre-selected amino acid of the firstmoiety.
 69. The method according to claim 68, wherein the pre-selectedamino acid is at the N-terminus or C-terminus of the first moiety. 70.The method according to claim 68, wherein the pre-selected amino acid isinternal to the first moiety.
 71. The method according to any one ofclaims 68 to 70, wherein the pre-selected amino acid is a non-naturalamino acid.
 72. The method according to any one of claims 66 to 71,wherein the first moiety is an antibody.
 73. The method according to anyone of claims 66 to 72, wherein the second moiety is as defined in anyone of claims 27 to
 35. 74. The method according to any one of claims 66to 72, wherein the second moiety is as defined in any one of claims 40to
 50. 75. The method according to any one of claims 66 to 74, whereinthe conjugating is by alkyne-azide cycloaddition.
 76. A method ofdegrading a cell surface molecule or extracellular molecule, comprising:contacting the cell surface molecule or extracellular molecule with thebifunctional molecule of any one of claims 1 to 61 under conditions inwhich the lysosomal targeting molecule shuttles the cell surfacemolecule or extracellular molecule to the lysosome for degradation. 77.The method according to claim 76, wherein the bifunctional moleculeenhances degradation of the cell surface molecule or extracellularmolecule relative to degradation of the cell surface molecule orextracellular molecule in the presence of the first moiety alone. 78.The method according to claim 76 or claim 77, wherein the method isperformed in vitro.
 79. The method according to claim 76 or claim 77,wherein the method is performed in vivo.
 80. A pharmaceuticalcomposition comprising: the bifunctional molecule of any one of claims 1to 61; and a pharmaceutically acceptable carrier.
 81. The pharmaceuticalcomposition of claim 80, wherein the composition is formulated forparenteral administration.
 82. A method comprising administering to anindividual in need thereof the pharmaceutical composition of claim 80 orclaim
 81. 83. A method of treating cancer comprising administering to anindividual having cancer an effective amount of the pharmaceuticalcomposition of claim 80 or claim
 81. 84. The method according to claim83, wherein the first moiety specifically binds to a molecule selectedfrom the group consisting of: a cell surface molecule on a cancer cell,a ligand for a cell surface molecule on a cancer cell, a cell surfacemolecule on an immune cell, a ligand for a cell surface molecule on animmune cell, an inhibitory immune receptor, and a ligand of aninhibitory immune receptor.
 85. The method according to claim 83 orclaim 84, wherein the individual has hepatocellular carcinoma (HCC), thefirst moiety binds a cell surface molecule on HCC cells of theindividual, and the second moiety binds ASGPR.
 86. The method accordingto claim 85, wherein the first moiety binds a growth factor on HCC cellsof the individual.
 87. The method according to claim 90, wherein thefirst moiety binds a growth factor selected from the group consistingof: EGFR, C-Met, IGF1R, and FGFR4.
 88. The method according to any oneof claims 85 to 87, wherein the second moiety is as defined in any oneof claims 36 to
 50. 89. A method of enhancing antibody-dependentcellular cytotoxicity (ADCC) comprising administering to an individualin need of ADCC the pharmaceutical composition of claim 80 or claim 81.90. A method of enhancing immunogenicity of a cancer in an individual,comprising administering to the individual the pharmaceuticalcomposition of claim 80 or claim
 81. 91. The method according to claim89 or claim 90, wherein the first moiety specifically binds to amolecule selected from the group consisting of: an inhibitory immunereceptor, and a ligand of an inhibitory immune receptor.
 92. The methodaccording to any one of claims 82 to 91, wherein the administering is byparenteral administration.
 93. The method according to any one of claims82 to 91, wherein the bifunctional molecule enhances degradation of thecell surface molecule or extracellular molecule relative to degradationof the cell surface molecule or extracellular molecule in the presenceof the first moiety alone.
 94. A kit comprising: the bifunctionalmolecule of any one of claims 1 to 61; and instructions for degradingthe cell surface molecule or extracellular molecule to which the firstmoiety specifically binds.
 95. The kit of claim 94, wherein theinstructions are for degrading the cell surface molecule orextracellular molecule in vitro.
 96. The kit of claim 94, wherein theinstructions are for degrading the cell surface molecule orextracellular molecule in vivo.
 97. A kit comprising: the bifunctionalmolecule of any one of claims 1 to 61 or the pharmaceutical compositionof claim 80 or claim 81; and instructions for administering thebifunctional molecule or pharmaceutical composition to an individual inneed thereof.
 98. The kit of claim 97, wherein the bifunctional moleculeor pharmaceutical composition is present in one or more unit dosages.99. The kit of claim 97, wherein the bifunctional molecule orpharmaceutical composition is present in two or more unit dosages. 100.A glycopolymer comprising: a polymer scaffold; and one or moremannose-6-phosphate receptor (M6PR) ligands attached to the polymerscaffold.
 101. The glycopolymer of claim 100, wherein the glycopolymeris a glycoprotein comprising one or more amino acids functionalized withthe one or more M6PR ligands.
 102. The glycopolymer of claim 101,wherein the glycoprotein is a N-carboxyanhydride (NCA)-derivedglycoprotein.
 103. The glycopolymer of any one of claims 100 to 102,wherein the one or more M6PR ligands comprise one or moremannose-6-phosphates (M6P).
 104. The glycopolymer of any one of claims100 to 103, wherein the one or more M6PR ligands comprise one or moreM6P analogs.
 105. The glycopolymer of claim 104, wherein the one or moreM6P analogs comprise one or more mannose-6-phosphonates (M6Pn).
 106. Theglycopolymer of any one of claims 100 to 105, wherein the polymerscaffold comprises from 1 to 500 M6PR ligands.
 107. A method of makingthe glycopolymer of any one of claims 100 to 106, comprising: attachingthe one or more M6PR ligands to the polymer scaffold; or synthesizingthe polymer scaffold from monomers functionalized with the one or moreM6PR ligands.
 108. The method according to claim 107, wherein thescaffold is polymerized from one or more monomers functionalized withthe one or more M6PR ligands, and wherein the synthesizing is bysolid-phase synthesis.
 109. The method according to claim 108, whereinthe glycopolymer is a glycoprotein polymer, and wherein the synthesizingis by solid-phase peptide synthesis.
 110. A kit comprising: theglycopolymer of any one of claims 100 to 106; and instructions forconjugating the glycopolymer to a molecule of interest.
 111. The kit ofclaim 110, further comprising reagents for conjugating the glycopolymerto a molecule of interest.
 112. The kit of claim 110 or claim 111,wherein the molecule of interest is a polypeptide.
 113. The kit of claim112, wherein the polypeptide is an antibody.
 114. The kit of any one ofclaims 110 to 113, wherein the molecule of interest specifically binds acell surface molecule or extracellular molecule.
 115. A monomerfunctionalized with one or more mannose-6-phosphate receptor (M6PR)ligands.
 116. The monomer of claim 115, wherein the monomer is an aminoacid.
 117. The monomer of claim 115, wherein the monomer is anon-natural amino acid.
 118. The monomer of any one of claims 115 to117, wherein the one or more M6PR ligands comprise one or moremannose-6-phosphates (M6P).
 119. The monomer of any one of claims 115 to118, wherein the one or more M6PR ligands comprise one or more M6Panalogs.
 120. The monomer of claim 119, wherein the one or more M6Panalogs comprise one or more mannose-6-phosphonates (M6Pn).